Method of cloning porcine animals

ABSTRACT

The present invention relates to materials and methods for cloning porcine animals. The invention relates in part to totipotent cells useful for cloning porcine animals, porcine embryos produced from such cells by employing nuclear transfer techniques, and porcine animals that arise from such cells and embryos.

FIELD OF THE INVENTION

The invention relates to the cloning of porcine animals.

BACKGROUND OF THE INVENTION

The following discussion of the background of the invention is merelyprovided to aid the reader in understanding the invention and is notadmitted to describe or constitute prior art to the present invention.

Researchers have been developing methods for cloning mammalian animalsover the past two decades. Some reported methods include the steps of(1) isolating a cell, most often an embryonic cell; (2) inserting thatcell or a nucleus isolated from the cell into an enucleated oocyte(e.g., the nucleus of the oocyte was previously extracted), and (3)allowing the embryo to mature in vivo.

The first successful nuclear transfer experiment using mammalian cellswas reported in 1983, where pronuclei isolated from a murine (mouse)zygote were inserted into an enucleated oocyte and resulted in liveoffspring(s). McGrath & Solter, 1983, Science 220:1300-1302.Subsequently, others described the production of chimeric murine embryos(e.g., embryos that contain a subset of cells having significantlydifferent nuclear DNA from other cells in the embryo) using murineprimordial germ cells (PGCs). These cells are and can give rise topluripotent cells. Matsui et al., 1992, Cell 70:841-847 and Resnick etal., 1992, Nature 359:550; Kato et al., 1994, Journal of Reproductionand Fertility Abstract Series, Society For the Study of Fertility,Annual Conference, Southampton, 13:38. In 1998, researchers reportedthat murine cumulus cells can be used as nuclear donors in cloningtechniques for establishing cloned murine animals. Wakayama et al.,1998, Nature 394: 369-374.

Another nuclear transfer experiment was reported in 1986, where an ovine(sheep) embryonic cell was used as a nuclear donor in a cloning processthat resulted in a cloned lamb. Willadsen, 1986, Nature 320:63-65. Morerecently, other lambs were reported to be cloned from ovine embryoniccells; serum deprived somatic cells; cells isolated from embryonicdiscs; and somatic mammary tissue. Campbell et al., 1996, Nature380:64-66; PCT Publication WO 95/20042; Wilmut et al., 1997, Nature385:810-813; and PCT Publications WO 96/07732 and WO 97/07669. Otherapproaches for cloning ovine animals involved manipulating theactivation state of an in vivo matured oocyte after nuclear transfer.PCT Publication WO 97/07668. Publications that disclose cloned lambsreport a cloning efficiency that is, at best, approximately 0.4%.Cloning efficiency, as calculated for the previous estimate, is a ratioequal to the number of cloned lambs divided by the number of nucleartransfers used to produce that number of cloned lambs.

Yet another nuclear transfer experiment resulted in a cloned bovineanimal (cattle), where the animal was cloned using an embryonic cellderived from a 2-64 cell embryo as a nuclear donor. This bovine animalwas reportedly cloned by utilizing nuclear transfer techniques set forthin U.S. Pat. No. 4,994,384 and 5,057,420. Others reported that clonedbovine embryos were formed where an inner cell mass cell of a blastocyststage embryo was utilized as a nuclear donor in a nuclear transferprocedure. Sims & First, 1993, Theriogenology 39:313 and Keefer et al.,1994, Mol. Reprod. Dev. 38:264-268. In addition, another publicationreported that cloned bovine embryos were prepared by nuclear transfertechniques that utilized a PGC isolated from fetal tissue as a nucleardonor. Delhaise et al., 1995, Reprod Fert. Develop. 7:1217-1219; Lavoir1994, J. Reprod Dev. 37:413-424; and PCT application WO 95/10599entitled “Embryonic Stem Cell-Like Cells.”

With regard to porcine animals (swine), researchers have reportedmethods for obtaining chimeric animals, specifically, where a nucleardonor is placed inside an enucleated embryonic cell. Prather et al.,1989, Biology of Reproduction 41: 414-418; Piedrahita et al., 1998,Biology of Reproduction 58: 1321-1329; and WO 94/26884, “Embryonic StemCells for Making Chimeric and Transgenic Ungulates,” Wheeler, publishedNov. 24, 1994.

Also, researchers have reported nuclear transfer experiments for porcinenuclear donors and porcine oocytes. See., e.g., Nagashima et al., 1997,Mol. Reprod. Dev. 48: 339-343; Nagashima et al., 1992, J. Reprod. Dev.38: 73-78; Prather et al., 1989, Biol. Reprod. 41: 414-419; Prather etal., 1990, Exp. Zool. 255: 355-358; Saito et al., 1992, Assis. Reprod.Tech. Andro. 259: 257-266; and Terlouw et al., 1992, Theriogenology 37:309.

In addition, researchers have reported methods for activating porcineoocytes. Grocholová et al., 1997, J. Exp. Zoology 277: 49-56; Schoenbecket al., 1993, Theriogenology 40: 257-266; Prather et al., 1991,Molecular Reproduction and Development 28: 405-409; Jolliff & Prather,1997, Biol. Reprod. 56: 544-548; Mattioli et al., 1991, MolecularReproduction and Development 30: 109-125; Terlouw et al., 1992,Theriogenology 37. 309; Prochazka et al., 1992, J. Reprod. Fert. 96:725-734; Funahashi et al., 1993, Molecular Reproduction and Development36: 361-367; Prather et al., Bio. Rep. Vol. 50 Sup 1. 282; Nussbaum etal., 1995, Molecular Reproduction and Development 41: 70-75; Funahashiet al., 1995, Zygote 3: 273-281; Wang et al., 1997, Biology ofReproduction 56: 1376-1382; Piedrahita et al., 1989, Biology ofReproduction 58: 1321-1329; Macháty et al., 1997, Biology ofReproduction 57: 85-91; and Macháty et al., 1995, Biology ofReproduction 52: 753-758.

There remains a long felt need for materials and methods that yieldefficient nuclear transfer using a porcine nuclear donor. This long feltneed is based in part upon a potential medical application, known asxenotransplantation, which includes procedures for extracting organsfrom porcine animals and transplanting these organs into humans in needof such organs. U.S. Pat. No. 5,589,582, Hawley et al., issued Dec. 31,1991; PCT application WO 95/28412, Baetsher et al., published Oct. 26,1995; PCT application WO 96/06165, Sachs et al., published Feb. 29,1996; PCT application WO 93/16729, Bazin, published Sep. 2, 1993; PCTapplication WO 97/12035, Diamond et al., published Apr. 3, 1997; PCTapplication WO 98/16630, Piedrahita & Bazer, published Apr. 23, 1998.

SUMMARY

The invention relates in part to cloning technologies for porcineanimals. The invention also relates in part to totipotent cells andcells that can be made totipotent, for use in cloning procedures andproduction of porcine animals, embryos produced from these porcine cellsusing nuclear transfer techniques, porcine animals that arise from thesecells and embryos, and methods and processes for establishing suchcells, embryos, and animals.

The present invention provides multiple advantages over tools andmethods currently utilized for porcine cloning. Such features andadvantages include:

(1) Production of cloned porcine animals from virtually any type ofcell. The invention provides materials and methods for reprogrammingnon-totipotent porcine cells into totipotent porcine cells. Thesenon-totipotent porcine cells may be of nonembryonic origin. This featureof the invention allows for an ability to assess a phenotype of anexisting porcine animal and then readily establish a totipotent cellline for cloning that animal.

(2) Establishment of totipotent porcine cell lines from virtually anytype of porcine cell. In one aspect of the invention, non-totipotentporcine precursor cells can be reprogrammed into totipotent cells. Thesenon-totipotent precursor cells may be non-embryonic cells. Establishedtotipotent porcine cell lines provide an advantage of enhancing cloningefficiency due to lower cellular heterogeneity within cell lines (e.g.,cell lines often exhibit lower rates of differentiation than primarycells). In addition, the totipotent cell lines can be manipulated invitro to produce porcine cells, embryos, and animals whose genomes havebeen manipulated (e.g., transgenic). Furthermore, totipotent cell linescan be more easily stored, transported, and re-established in culturethan other types of cell lines.

(3) Efficiency enhancement for cloning embryos as a result of utilizingasynchronous and karyotypically stable porcine cell lines in a completein vitro embryo production system.

Cloning efficiency can be expressed by the ratio between the number ofembryos resulting from nuclear transfer and the number of nucleartransfers performed to give rise to the embryos. Alternatively, cloningefficiency can be expressed as the ratio between the number of live bornanimals and the number of nuclear transfers performed to give rise tothese animals.

Cultured Cells of the Invention

In a first aspect, the invention features a totipotent porcine cell.

The term “porcine” as used herein can refer to any animal of the familySuidae. A porcine animal can refer to swine of any sort, including, butnot limited to, wild boar, domestic swine, miniswine, warthog, peccary,and barboosa. For examples of miniswine, see, e.g., Bustad & McClellan,1968, Lab. Anim. Care. 18: 280-287 and England & Panepinto, 1986,“Conceptual and operational history of the development of miniatureswine,” Swine in Biomedical Research (M. E. Tubleson, ed.), PlenumPress, N Y pp 17-22, each of which is incorporated herein by referencein its entirety, including all figures, tables, and drawings.

The term “totipotent” as used herein can refer to a cell that gives riseto a live born animal. The term “totipotent” can also refer to a cellthat gives rise to all of the cells in a particular animal. A totipotentcell can give rise to all of the cells of an animal when it is utilizedin a procedure for developing an embryo from one or more nucleartransfer steps. Totipotent cells may also be used to generate incompleteanimals such as those useful for organ harvesting, e.g., having geneticmodifications to eliminate growth of an organ or appendage bymanipulation of a homeotic gene.

The term “live born” as used herein preferably refers to an animal thatexists ex utero. A “live born” animal may be an animal that is alive forat least one second from the time it exits the maternal host. A “liveborn” animal may not require the circulatory system of an in uteroenvironment for survival. A “live born” animal may be an ambulatoryanimal. Such animals can include pre- and post-pubertal animals. Asdiscussed previously, a live born animal may lack a portion of whatexists in a normal animal of its kind.

In preferred embodiments, totipotent cells are (1) cultured; (2) arecultured as cell lines; and are (3) cultured as permanent cell lines.

The term “cultured” as used herein in reference to cells can refer toone or more cells that are undergoing cell division or not undergoingcell division in an in vitro environment. An in vitro environment can beany medium known in the art that is suitable for maintaining cells invitro, such as suitable liquid media or agar, for example. Specificexamples of suitable in vitro environments for cell cultures aredescribed in Culture of Animal Cells: a manual of basic techniques(3^(rd) edition), 1994, R. I. Freshney (ed.), Wiley-Liss, Inc.; Cells: alaboratory manual (vol. 1), 1998, D. L. Spector, R. D. Goldman, L. A.Leinwand (eds.), Cold Spring Harbor Laboratory Press; and Animal Cells:culture and media, 1994, D. C. Darling, S. J. MorganJohn Wiley and Sons,Ltd., each of which is incorporated herein by reference in its entiretyincluding all figures, tables, and drawings. Cells may be cultured insuspension and/or in monolayers with one or more substantially similarcells. Cells may be cultured in suspension and/or in monolayers with aheterogeneous population of cells. The term “heterogeneous” as utilizedin the previous sentence can relate to any cell characteristics, such ascell type and cell cycle stage, for example. Cells may be cultured insuspension, cultured as monolayers attached to a solid support, and/orcultured on a layer of feeder cells, for example. The term “feedercells” is defined hereafter. Furthermore, cells may be successfullycultured by plating the cells in conditions where they lack cell to cellcontact. Preferably, cultured cells undergo cell division and arecultured for at least 5 days, more preferably for at least 10 days or 20days, and most preferably for at least 30 days. Preferably, asignificant number of cultured cells do not terminate while in culture.The terms “terminate” and “significant number are defined” hereafter.Nearly any type of cell can be placed in cell culture conditions.Cultured cells can be utilized to establish a cell line.

The term “cell line” as used herein can refer to cultured cells that canbe passaged at least one time without terminating. The invention relatesto cell lines that can be passaged at least 1, 2, 5, 10, 15, 20, 30, 40,50, 60, 80, 100, and 200 times. Cell passaging is defined hereafter.

The term “terminating” and “terminate” as used herein with regard tocultured cells may refer to cells that undergo cell death, which can bemeasured using multiple techniques known to those skilled in the art(e.g., CytoTox96® Cytotoxicity Assay, Promega, Inc. catalog no. G1780;Celltiter96® Aqueous Cell Proliferation Assay Kit, Promega, Inc. catalogno. G3580; and Trypan Blue solution for cytotoxicity assays, Sigmacatalog no. T6146). Termination may also be a result of apoptosis, whichcan be measured using multiple techniques known to persons skilled inthe art (e.g., Dead End™ Apoptosis Detection Kit, Promega, Inc. catalogno. G7130). Terminated cells may be identified as those that haveundergone cell death and/or apoptosis and have released from a solidsurface in culture. In addition, terminated cells may lack intactmembranes which can be identified by procedures described above. Also,terminated cells may exhibit decreased metabolic activity, which may becaused in part by decreased mitochondrial activity that can beidentified by rhodamine 1,2,3, for example. Furthermore, termination canbe refer to cell cultures where a significant number of cultured cellsterminate. The term “significant number” in the preceding sentence canrefer to about 80% of the cells in culture, preferably about 90% of thecells in culture, more preferably about 100% of the cells in culture,and most preferably 100% of the cells in culture.

The term “suspension” as used herein can refer to cell cultureconditions in which cells are not attached to a solid support. Cellsproliferating in suspension can be stirred while proliferating usingapparatus well known to those skilled in the art.

The term “monolayer” as used herein can refer to cells that are attachedto a solid support while proliferating in suitable culture conditions. Asmall portion of cells proliferating in a monolayer under suitablegrowth conditions may be attached to cells in the monolayer but not tothe solid support. Preferably less than 15% of these cells are notattached to the solid support, more preferably less than 10% of thesecells are not attached to the solid support, and most preferably lessthan 5% of these cells are not attached to the solid support.

The term “substantially similar” as used herein in reference to porcinecells can refer to cells from the same organism and the same tissue. Theterm “substantially similar” can also refer to cell populations thathave not significantly differentiated. For example, preferably less than15% of the cells in a population of cells have differentiated, morepreferably less than 10% of the cell population have differentiated, andmost preferably less than 5% of the cell population have differentiated.

The term “plated” or “plating” as used herein in reference to cells canrefer to establishing cell cultures in vitro. For example, cells can bediluted in cell culture media and then added to a cell culture plate,dish, or flask. Cell culture plates are commonly known to a person ofordinary skill in the art. Cells may be plated at a variety ofconcentrations and/or cell densities.

The term “cell plating” can also extend to the term “cell passaging.”Cells of the invention can be passaged using cell culture techniqueswell known to those skilled in the art. The term “cell passaging” canrefer to a technique that involves the steps of (1) releasing cells froma solid support or substrate and disassociation of these cells, and (2)diluting the cells in media suitable for further cell proliferation.Cell passaging may also refer to removing a portion of liquid mediumcontaining cultured cells and adding liquid medium to the originalculture vessel to dilute the cells and allow further cell proliferation.In addition, cells may also be added to a new culture vessel which hasbeen supplemented with medium suitable for further cell proliferation.

The term “proliferation” as used herein in reference to cells can referto a group of cells that can increase in number over a period of time.

The term “confluence” as used herein can refer to a group of cells wherea large percentage of cells are physically contacted with at least oneother cell in that group. Confluence may also be defined as a group ofcells that grow to a maximum cell density in the conditions provided.For example, if a group of cells can proliferate in a monolayer and theyare placed in a culture vessel in a suitable growth medium, they areconfluent when the monolayer has spread across a significant surfacearea of the culture vessel. The surface area covered by the cellspreferably represents about 50% of the total surface area, morepreferably represents about 70% of the total surface area, and mostpreferably represents about 90% of the total surface area.

The term “permanent” or “immortalized” as used herein in reference toporcine cells can refer to cells that may undergo cell division anddouble in cell numbers while cultured in an in vitro environment amultiple number of times until the cells terminate. A permanent cellline may double over 10 times before a significant number of cellsterminate in culture. Preferably, a permanent cell line may double over20 times or over 30 times before a significant number of cells terminatein culture. More preferably, a permanent cell line may double over 40times or 50 times before a significant number of cells terminate inculture. Most preferably, a permanent cell line may double over 60 timesbefore a significant number of cells terminate in culture. The term“terminate” is described previously. Cell doubling can be measured bycounting the number of cells in culture using techniques well known to aperson of ordinary skill in the art. As a measure of cell culturepermanence, a number of doublings can be measured until a significantnumber of cells terminate in culture. The term “significant number” isalso described previously.

Permanent cells may be distinguished from non-permanent cells on thebasis that permanent cells can be passaged at densities lower than thoseof non-permanent cells. Specifically, permanent cells can be grown toconfluence (described hereafter) when plating conditions do not allowphysical contact between the cells. Hence, permanent cells can bedistinguished from non-permanent cells when cells are plated at celldensities where the cells do not physically contact one another.

In preferred embodiments, (1) totipotent cells are not alkalinephosphatase positive (e.g., cells do not appreciably stain for alkalinephosphatase); (2) totipotent cells arise from at least one precursorcell; (3) a precursor cell is isolated from and/or arises from anyregion of a porcine animal; (4) a precursor cell is isolated from and/orarises from any cell in culture; (5) a precursor cell is selected fromthe group consisting of a non-embryonic cell, a non-fetal cell, adifferentiated cell, an undifferentiated cell, a somatic cell, anembryonic cell, a fetal cell, an embryonic stem cell, a primordial germcell, a genital ridge cell, a cumulus cell, an amniotic cell, a fetalfibroblast cell, a hepatacyte, an embryonic germ cell, an adult cell, acell isolated from an asynchronous population of cells, and a cellisolated from a synchronized population of cells where the synchronouspopulation is not arrested in the G₀ stage of the cell cycle; (6)totipotent cells have a morphology of an embryonic germ cell.

The term “alkaline phosphatase positive” as used herein can refer to adetectable presence of cellular alkaline phosphatase. Cells that are notalkaline phosphatase positive do not stain appreciably using a procedurefor visualizing cellular alkaline phosphatase. Procedures for detectingthe presence of cellular alkaline phosphatase are well-known to a personof ordinary skill in the art. See, e.g., Matsui et al., 1991, “Effect ofSteel Factor and Leukemia Inhibitory Factor on Murine Primordial GermCells in Culture,” Nature 353: 750-752. Examples of cells that stainappreciably for alkaline phosphatase can be found in the art. See, e.g.,U.S. Pat. No. 5,453,357, Entitled “Pluripotent Embryonic Stem Cells andMethods of Making Same,” issued to Hogan on Sep. 26, 1995, which isincorporated by reference herein in its entirety, including all figures,tables, and drawings.

The term “precursor cell” or “precursor cells” as used herein can referto a cell or cells used to establish cultured porcine cells or acultured porcine cell line. A precursor cell or cells may be isolatedfrom nearly any cellular entity. For example, a precursor cell or cellsmay be isolated from blastocysts, embryos, fetuses, and cell lines(e.g., cell lines established from embryonic cells), preferably isolatedfrom fetuses and/or cell lines established from fetal cells, and morepreferably isolated from ex utero animals and/or cell cultures and/orcell lines established from such ex utero animals. An ex utero animalmay exist as a newborn animal (e.g., 5 days after birth), adolescentanimal (e.g., pre-pubescent animal), pubescent animal (e.g., afterovulation or production of viable sperm), and adult animal (e.g., postpubescent). The ex utero animals may be alive or post mortem. Precursorcells may be cultured or non-cultured. Furthermore, precursor cells maybe at a time cryopreserved or frozen (e.g., cryopreserved cells may beutilized as precursor cells to establish a cell culture). These examplesare not meant to be limiting and a further description of theseexemplary precursor cells is provided hereafter.

The term “arises from” as used herein can refer to the conversion of oneor more cells into one or more cells having at least one differingcharacteristic. For example, (1) a non-totipotent precursor cell can beconverted into a totipotent cell by utilizing features of the inventiondescribed hereafter; (2) a precursor cell can develop a cell morphologyof an embryonic germ cell; (3) a precursor cell can give rise to acultured cell; (4) a precursor cell can give rise to a cultured cellline; and (5) a precursor cell can give rise to a cultured permanentcell line. A conversion process can be referred to as a reprogrammingstep. In addition, the term “arises from” can refer to establishingtotipotent embryos from totipotent cells of the invention by using anuclear transfer process, as described hereafter.

The term “reprogramming” or “reprogrammed” as used herein can refer tomaterials and methods that can convert a cell into another cell havingat least one differing characteristic. Also, such materials and methodsmay reprogram or convert a cell into another cell type that is nottypically expressed during the life cycle of the former cell. Forexample, (1) a non-totipotent cell can be reprogrammed into antotipotent cell; (2) a precursor cell can be reprogrammed into a cellhaving a morphology of an EG cell; and (3) a precursor cell can bereprogrammed into a totipotent cell. An example of materials and methodsfor converting a precursor cell into a totipotent cell having EG cellmorphology is described hereafter.

The term “isolated” as used herein can refer to a cell that ismechanically separated from another group of cells. Examples of a groupof cells are a developing cell mass, a cell culture, a cell line, and ananimal. These examples are not meant to be limiting and the inventionrelates to any group of cells. Methods for isolating one or more cellsfrom another group of cells are well known in the art. See, e.g.,Culture of Animal Cells: a manual of basic techniques (3^(rd) edition),1994, R. I. Freshney (ed.), Wiley-Liss, Inc.; Cells: a laboratory manual(vol. 1), 1998, D. L. Spector, R. D. Goldman, L. A. Leinwand (eds.),Cold Spring Harbor Laboratory Press; and Animal Cells: culture andmedia, 1994, D. C. Darling, S. J. Morgan John Wiley and Sons, Ltd.

The term “non-embryonic cell” as used herein can refer to a cell that isnot isolated from an embryo. Non-embryonic cells can be differentiatedor nondifferentiated. Non-embryonic cells can refer to nearly anysomatic cell, such as cells isolated from an ex utero animal. Theseexamples are not meant to be limiting.

For the purposes of the present invention, the term “embryo” or“embryonic” as used herein can refer to a developing cell mass that hasnot implanted into an uterine membrane of a maternal host. Hence, theterm “embryo” as used herein can refer to a fertilized oocyte, a cybrid(defined herein), a pre-blastocyst stage developing cell mass, and/orany other developing cell mass that is at a stage of development priorto implantation into an uterine membrane of a maternal host. Embryos ofthe invention may not display a genital ridge. Hence, an “embryoniccell” is isolated from and/or has arisen from an embryo.

An embryo can represent multiple stages of cell development. Forexample, a one cell embryo can be referred to as a zygote, a solidspherical mass of cells resulting from a cleaved embryo can be referredto as a morula, and an embryo having a blastocoel can be referred to asa blastocyst.

The term “fetus” as used herein can refer to a developing cell mass thathas implanted into the uterine membrane of a maternal host. A fetus caninclude such defining features as a genital ridge, for example. Agenital ridge is a feature easily identified by a person of ordinaryskill in the art, and is a recognizable feature in fetuses of mostanimal species. The term “fetal cell” as used herein can refer to anycell isolated from and/or has arisen from a fetus or derived from afetus, including amniotic cells. The term “non-fetal cell” is a cellthat is not derived or isolated from a fetus.

When precursor cells are isolate from a fetus, such precursor cells arepreferably isolated from porcine fetuses where the fetus is between 20days and parturition, between 30 days and 100 days, more preferablybetween 35 days and 70 days and between 40 days and 60 days, and mostpreferably about a 55 day fetus. An age of a fetus can be determined bythe time that an embryo, which develops into the fetus, is established.For example, a two cell embryo can be referred to as a day one embryothat can develop into a 54 day fetus. The term “about” with respect tofetuses can refer to plus or minus five days.

The term “parturition” as used herein can refer to a time that a fetusis delivered from female recipient. A fetus can be delivered from afemale recipient by abortion, c-section, or birth.

The term “primordial germ cell” as used herein can refer to a diploidprecursor cell capable of becoming a germ cell. Primordial germ cellscan be isolated from any tissue in a developing cell mass, and arepreferably isolated from genital ridge cells of a developing cell mass.A genital ridge is a section of a developing cell mass that iswell-known to a person of ordinary skill in the art. See, e.g.,Strelchenko, 1996, Theriogenology 45: 130-141 and Lavoir 1994, J.Reprod. Dev. 37: 413-424.

The term “embryonic stem cell” as used herein can refer to pluripotentcells isolated from an embryo that are maintained in in vitro cellculture. Embryonic stem cells may be cultured with or without feedercells. Embryonic stem cells can be established from embryonic cellsisolated from embryos at any stage of development, including blastocyststage embryos and pre-blastocyst stage embryos. Embryonic stem cells mayhave a rounded cell morphology and may grow in rounded cell clumps onfeeder layers. Embryonic stem cells are well known to a person ofordinary skill in the art. See, e.g., WO 97/37009, entitled “CulturedInner Cell Mass Cell-Lines Derived from Ungulate Embryos,” Stice andGolueke, published Oct. 9, 1997, and Yang & Anderson, 1992,Theriogenology 38: 315-335, each of which is incorporated herein byreference in its entirety, including all figures, tables, and drawings.See, e.g., Piedrahita et al., 1998, Biol. Reprod. 58: 1321-1329; Wiannyet al., 1997, Biol. Reprod. 57: 756-764; Moore & Piedrahita, 1997, InVitro Cell Biol. Anim. 33: 62-71; Moore, & Piedrahita, 1996, Mol.Reprod. Dev. 45: 139-144; Wheeler, 1994, Reprod. Fert. Dev. 6: 563-568;Hochereau-de Reviers & Perreau, Reprod. Nutr. Dev. 33: 475-493; Strojeket al., 1990, Theriogenology 33: 901-903; Piedrahita et al., 1990,Theriogenology 34: 879-901; and Evans et al., 1990, Theriogenology 33:125-129, each of which is incorporated herein by reference in itsentirety, including all figures, tables, and drawings.

The term “differentiated cell” as used herein can refer to a precursorcell that has developed from an unspecialized phenotype to a specializedphenotype. For example, embryonic cells can differentiate into anepithelial cell lining the intestine. Materials and methods of theinvention can reprogram differentiated cells into totipotent cells.Differentiated cells can be isolated from a fetus or a live born animal,for example.

The term “undifferentiated cell” as used herein can refer to a precursorcell that has an unspecialized phenotype and is capable ofdifferentiating. An example of an undifferentiated cell is a stem cell.

The term “asynchronous population” as used herein can refer to cellsthat are not arrested at any one stage of the cell cycle. Many cells canprogress through the cell cycle and do not arrest at any one stage,while some cells can become arrested at one stage of the cell cycle fora period of time. Some known stages of the cell cycle are G₁, S, G₂, andM. An asynchronous population of cells is not manipulated to synchronizeinto any one or predominantly into any one of these phases. Cells can bearrested in the M stage of the cell cycle, for example, by utilizingmultiple techniques known in the art, such as by colcemid exposure.Examples of methods for arresting cells in one stage of a cell cycle arediscussed in WO 97/07669, entitled “Quiescent Cell Populations forNuclear Transfer,” hereby incorporated herein by reference in itsentirety, including all figures, tables, and drawings.

The terms “synchronous population” and “synchronizing” as used hereincan refer to a fraction of cells in a population that are within a samestage of the cell cycle. Preferably, about 50% of cells in a populationof cells are arrested in one stage of the cell cycle, more preferablyabout 70% of cells in a population of cells are arrested in one stage ofthe cell cycle, and most preferably about 90% of cells in a populationof cells are arrested in one stage of the cell cycle. Cell cycle stagecan be distinguished by relative cell size as well as by a variety ofcell markers well known to a person of ordinary skill in the art. Forexample, cells can be distinguished by such markers by using flowcytometry techniques well known to a person of ordinary skill in theart. Alternatively, cells can be distinguished by size utilizingtechniques well known to a person of ordinary skill in the art, such asby the utilization of a light microscope and a micrometer, for example.In a preferred embodiment, cells are synchronized by arresting them(i.e., cells are not dividing) in a discreet stage of the cell cycle.

The terms “embryonic germ cell” and “EG cell” as used herein can referto a cultured cell that has a distinct flattened morphology and can growwithin monolayers in culture. An EG cell may be distinct from afibroblast cell. This EG cell morphology is to be contrasted with cellsthat have a spherical morphology and form multicellular clumps on feederlayers. Porcine embryonic germ cells may not require the presence offeeder layers or presence of growth factors in cell culture conditions.Porcine embryonic germ cells may also grow with decreased doubling rateswhen these cells approach confluence on culture plates. Porcineembryonic germ cells of the invention may be totipotent. Porcineembryonic germ cells of the invention may not appreciably stain foralkaline phosphatase. Preferably, porcine embryonic germ cells areestablished in culture media that contains a significant concentrationof glucose, as described herein.

Porcine embryonic germ cells may be established from a cell culture ofnearly any type of porcine precursor cell. Examples of precursor cellsare discussed herein, and a preferred precursor cell for establishing aporcine embryonic germ cell culture is a genital ridge cell from afetus. Genital ridge cells are preferably isolated from porcine fetuseswhere the fetus is between 20 days and parturition, between 30 days and100 days, more preferably between 35 days and 70 days and between 40days and 60 days, and most preferably about a 55 day fetus. An age of afetus can be determined as described above. The term “about” withrespect to fetuses can refer to plus or minus five days. As describedherein, EG cells may be physically isolated from a primary culture ofcells, and these isolated EG cells may be utilized to establish a cellculture that eventually forms a homogenous or nearly homogenous cellline of EG cells.

The terms “morphology” and “cell morphology” as used herein can refer toform, structure, and physical characteristics of cells. For example, onecell morphology is significant levels of alkaline phosphatase, and thiscell morphology can be identified by determining whether a cell stainsappreciably for alkaline phosphatase. Another example of a cellmorphology is whether a cell is flat or round in appearance whencultured on a surface or in the presence of a layer of feeder cells.Many other cell morphologies are known to a person of ordinary skill inthe art and are cell morphologies are readily identifiable usingmaterials and methods well known to those skilled in the art. See, e.g.,Culture of Animal Cells: a manual of basic techniques (3^(rd) edition),1994, R. I. Freshney (ed.), Wiley-Liss, Inc.

The term “cumulus cell” as used herein can refer to any cultured ornon-cultured cell that is isolated from cells and/or tissue surroundingan oocyte. Persons skilled in the art can readily identify a cumuluscell. Examples of methods for isolating and culturing cumulus cells arediscussed in Damiani et al., 1996, Mol. Reprod. Dev. 45: 521-534; Longet al., 1994, J. Reprod. Fert. 102: 361-369; and Wakayama et al., 1998,Nature 394: 369-373, each of which is incorporated herein by referencein its entireties, including all figures, tables, and drawings.

The term “amniotic cell” as used herein can refer to a cultured ornon-cultured cell isolated from amniotic fluid or tissues in contactwith amniotic fluid. Persons skilled in the art can readily identify anamniotic cell. Examples of methods for isolating and culturing amnioticcells are discussed in Bellow et al., 1996, Theriogenology 45: 225;Garcia & Salaheddine, 1997, Theriogenology 47: 1003-1008; Leibo & Rail,1990, Theriogenology 33: 531-552; and Vos et al., 1990, Vet. Rec. 127:502-504, each of which is incorporated herein by reference in itsentirety, including all figures tables and drawings.

The term “fetal fibroblast cell” as used herein can refer to anydifferentiated porcine fetal cell having a fibroblast appearance.Fibroblasts can have a flattened appearance when cultured on culturemedia plates. Fetal fibroblast cells can also have a spindle-likemorphology, density limited for growth, and can have a finite life spanin culture of approximately fifty generations. In addition, fetalfibroblast cells may rigidly maintain a diploid chromosomal content andmay generate type I collagen. For a description of fibroblast cells,see, e.g., Culture of Animal Cells: a manual of basic techniques (3^(rd)edition), 1994, R. I. Freshney (ed), Wiley-Liss, Inc

The term “adult cell” as used herein can refer to any cell isolated froman adult porcine animal. Such an adult cell can be isolated from anypart of the porcine animal, including, but not limited to, skin from anear, skin from an abdominal region, kidney, liver, heart, and lung.Procedures are set forth herein for culturing such adult cells.

In preferred embodiments, (1) totipotent porcine cells of the inventioncomprise modified nuclear DNA; (2) modified nuclear DNA includes a DNAsequence that encodes a recombinant product; (3) a recombinant productis a polypeptide; (4) a recombinant product is a ribozyme; (4) arecombinant product is expressed in a biological fluid or tissue; (5) arecombinant product confers or partially confers resistance to one ormore diseases; (6) a recombinant product confers resistance or partiallyconfers resistance to one or more parasites; (7) a modified nuclear DNAcomprises at least one other DNA sequence that can function as aregulatory element; (8) a regulatory element is selected from the groupconsisting of promoter, enhancer, insulator, and repressor; and (9) aregulatory element is selected from the group consisting of milk proteinpromoter, urine protein promoter, blood protein promoter, lacrimal ductprotein promoter, synovial protein promoter, mandibular gland proteinpromoter, casein promoter, β-casein promoter, melanocortin promoter,milk serum protein promoter, α-lactalbumin promoter, whey acid proteinpromoter, uroplakin promoter, α-actin promoter.

The term “modified nuclear DNA” as used herein can refer to a nucleardeoxyribonucleic acid sequence of a cell, embryo, fetus, or animal ofthe invention that has been manipulated by one or more recombinant DNAtechniques. Examples of recombinant DNA techniques are well known to aperson of ordinary skill in the art, which can include (1) inserting aDNA sequence from another organism (e.g., a human organism) into targetnuclear DNA, (2) deleting one or more DNA sequences from target nuclearDNA, and (3) introducing one or more base mutations (e.g., site-directedmutations) into target nuclear DNA. Cells with modified nuclear DNA canbe referred to as “transgenic cells” for the purposes of the invention.Transgenic cells can be useful as materials for nuclear transfer cloningtechniques provided herein.

Methods and tools for insertion, deletion, and mutation of nuclear DNAof mammalian cells are well-known to a person of ordinary skill in theart. See, Molecular Cloning, a Laboratory Manual, 2nd Ed., 1989,Sambrook, Fritsch, and Maniatis, Cold Spring Harbor Laboratory Press;U.S. Pat. No. 5,633,067, “Method of Producing a Transgenic Bovine orTransgenic Bovine Embryo,” DeBoer et al., issued May 27, 1997; U.S. Pat.No. 5,612,205, “Homologous Recombination in Mammalian Cells,” Kay etal., issued Mar. 18, 1997; and PCT publication WO 93/22432, “Method forIdentifying Transgenic Pre-Implantation Embryos”; WO 98/16630,Piedrahita & Bazer, published Apr. 23, 1998, “Methods for the Generationof Primordial Germ Cells and Transgenic Animal Species,” each of whichis incorporated herein by reference in its entirety, including allfigures, drawings, and tables. These methods include techniques fortransfecting cells with foreign DNA fragments and the proper design ofthe foreign DNA fragments such that they effect insertion, deletion,and/or mutation of the target DNA genome.

Transgenic cells may be obtained in a variety of manners. For example,transgenic cells can be isolated from a transgenic animal. Examples oftransgenic porcine animals are well known in the art. Cells isolatedfrom a transgenic animal can be converted into totipotent cells by usingthe materials and methods provided herein. In another example,transgenic cells can be established from totipotent cells of theinvention. Materials and methods for converting non-transgenic cellsinto transgenic cells are well known in the art, as describedpreviously.

Any of the cell types defined herein can be altered to harbor modifiednuclear DNA. For example, embryonic stem cells, embryonic germ cells,fetal cells, and any totipotent cell defined herein can be altered toharbor modified nuclear DNA.

Examples of methods for modifying a target DNA genome by insertion,deletion, and/or mutation are retroviral insertion, artificialchromosome techniques, gene insertion, random insertion with tissuespecific promoters, homologous recombination, gene targeting,transposable elements, and/or any other method for introducing foreignDNA. Other modification techniques well known to a person of ordinaryskill in the art include deleting DNA sequences from a genome, and/oraltering nuclear DNA sequences. Examples of techniques for alteringnuclear DNA sequences are site-directed mutagenesis and polymerase chainreaction procedures. Therefore, the invention relates in part to porcinecells that are simultaneously totipotent and transgenic. Such transgenicand totipotent cells can serve as nearly unlimited sources of donorcells for production of cloned transgenic porcine animals.

The term “recombinant product” as used herein can refer to the productproduced from a DNA sequence that comprises at least a portion of themodified nuclear DNA. This product can be a peptide, a polypeptide, aprotein, an enzyme, an antibody, an antibody fragment, a polypeptidethat binds to a regulatory element (a term described hereafter), astructural protein, an RNA molecule, and/or a ribozyme, for example.These products are well defined in the art. This list of products is forillustrative purposes only and the invention relates to other types ofproducts.

The term “ribozyme” as used herein can refer to ribonucleic acidmolecules that can cleave other RNA molecules in specific regions.Ribozymes can bind to discrete regions on a RNA molecule, and thenspecifically cleave a region within that binding region or adjacent tothe binding region. Ribozyme techniques can thereby decrease the amountof polypeptide translated from formerly intact message RNA molecules.For specific descriptions of ribozymes, see U.S. Pat. No. 5,354,855,entitled “RNA Ribozyme which Cleaves Substrate RNA without Formation ofa Covalent Bond,” Cech et al., issued on Oct. 11, 1994, and U.S. Pat.No. 5,591,610, entitled “RNA Ribozyme Polymerases, Dephosphorylases,Restriction Endoribonucleases and Methods,” Cech et al., issued on Jan.7, 1997, each of which is incorporated herein by reference in itsentirety including all figures, tables, and drawings.

The terms “biological fluid” or “tissue” as used herein can refer to anyfluid or tissue in a biological organism. Fluids may include, but arenot limited to, tears, saliva, milk, urine, amniotic fluid, semen,plasma, oviductal fluid, and synovial fluid. Tissues may include, butare not limited to, lung, heart, blood, liver, muscle, brain, pancreas,skin, and others.

The term “confers resistance” as used herein can refer to the ability ofa recombinant product to completely abrogate or partially alleviate thesymptoms of a disease or parasitic condition. Hence, if a disease isrelated to inflammation, for example, a recombinant product can conferresistance to that inflammation if inflammation decreases uponexpression of the recombinant product. A recombinant product may conferresistance or partially confer resistance to a disease or parasiticcondition, for example, if a recombinant product is an anti-sense RNAmolecule that specifically binds to an mRNA molecule encoding apolypeptide responsible for inflammation. Other examples of conferringresistance to diseases or parasites are described hereafter. Inaddition, examples of diseases are described hereafter.

Examples of parasites and strategies for conferring resistance to theseparasites are described hereafter. These examples include, but are notlimited to, worms, nematodes, insects, invertebrate, bacterial, viral,and eukaryotic parasites. These parasites can lead to diseased statesthat can be controlled by materials and methods of the invention.

The term “regulatory element” as used herein can refer to a DNA sequencethat can increase or decrease an amount of product produced from anotherDNA sequence. A regulatory element can cause the constitutive productionof the product (e.g., the product can be expressed constantly).Alternatively, a regulatory element can enhance or diminish productionof a recombinant product in an inducible fashion (e.g., the product canbe expressed in response to a specific signal). A regulatory element canbe controlled, for example, by nutrition, by light, or by adding asubstance to the transgenic organism's system. Examples of regulatoryelements well-known to those of ordinary skill in the art are promoters,enhancers, insulators, and repressors. See, e.g., Transgenic Animals,Generation and Use, 1997, Edited by L. M. Houdebine, Hardwood AcademicPublishers, Australia, hereby incorporated herein by reference in itsentirety including all figures, tables, and drawings.

The term “promoters” or “promoter” as used herein can refer to a DNAsequence that is located adjacent to a DNA sequence that encodes arecombinant product. A promoter is preferably linked operatively to anadjacent DNA sequence. A promoter typically increases an amount ofrecombinant product expressed from a DNA sequence as compared to anamount of the expressed recombinant product when no promoter exists. Apromoter from one organism specie can be utilized to enhance recombinantproduct expression from a DNA sequence that originates from anotherorganism specie. In addition, one promoter element can increase anamount of recombinant products expressed for multiple DNA sequencesattached in tandem. Hence, one promoter element can enhance theexpression of one or more recombinant products. Multiple promoterelements are well-known to persons of ordinary skill in the art.Examples of promoter elements are described hereafter.

The term “enhancers” or “enhancer” as used herein can refer to a DNAsequence that is located adjacent to the DNA sequence that encodes arecombinant product. Enhancer elements are typically located upstream ofa promoter element or can be located downstream of a coding DNA sequence(e.g., a DNA sequence transcribed or translated into a recombinantproduct or products). Hence, an enhancer element can be located 100 basepairs, 200 base pairs, or 300 or more base pairs upstream of a DNAsequence that encodes recombinant product. Enhancer elements canincrease an amount of recombinant product expressed from a DNA sequenceabove increased expression afforded by a promoter element. Multipleenhancer elements are readily available to persons of ordinary skill inthe art.

The term “insulators” or “insulator” as used herein can refer to DNAsequences that flank the DNA sequence encoding the recombinant product.Insulator elements can direct recombinant product expression to specifictissues in an organism. Multiple insulator elements are well known topersons of ordinary skill in the art. See, e.g., Geyer, 1997, Curr.Opin. Genet. Dev. 7: 242-248, hereby incorporated herein by reference inits entirety, including all figures, tables, and drawings.

The term “repressor” or “repressor element” as used herein can refer toa DNA sequence located in proximity to the DNA sequence that encodesrecombinant product, where a repressor sequence can decrease an amountof recombinant product expressed from that DNA sequence. Repressorelements can be controlled by binding of a specific molecule or specificmolecules to a repressor element DNA sequence. These molecules caneither activate or deactivate a repressor element. Multiple repressorelements are available to a person of ordinary skill in the art.

The terms “milk protein promoter,” “urine protein promoter,” “bloodprotein promoter,” “lacrimal duct protein promoter,” “synovial proteinpromoter,” and “mandibular gland protein promoter” refer to promoterelements that regulate the specific expression of proteins within thespecified fluid or gland or cell type in an animal. For example, a milkprotein promoter is a regulatory element that can control expression ofa protein that is expressed in milk of an animal. Other promoters, suchas casein promoter, α-lactalbumin promoter, whey acid protein promoter,uroplakin promoter, and α-actin promoter, for example, are well known toa person of ordinary skill in the art.

In preferred embodiments, (1) the totipotent porcine cell is subject tomanipulation; (2) the manipulation comprises the step of utilizing atotipotent porcine cell in a nuclear transfer procedure; (3) themanipulation comprises the step of cryopreserving totipotent cells; (4)the manipulation comprises the step of thawing totipotent cells; (5) themanipulation comprises the step of passaging totipotent cells; (6) themanipulation comprises the step of synchronizing totipotent cells; (7)the manipulation comprises the step of transfecting totipotent cellswith foreign DNA; and (8) the manipulation comprises the step ofdissociating a cell from another cell or group of cells.

The term “manipulation” as used herein can refer to common usage of theterm, which is management or handling directed towards some object.Examples of manipulations are described herein.

The term “nuclear transfer” as used herein can refer to introducing afull complement of nuclear DNA from one cell to an enucleated cell.Nuclear transfer methods are well known to a person of ordinary skill inthe art. See., e.g., Nagashima et al., 1997, Mol. Reprod. Dev. 48:339-343; Nagashima et al., 1992, J. Reprod. Dev. 38: 73-78; Prather etal., 1989, Biol. Reprod. 41: 414-419; Prather et al., 1990, Exp. Zool.255: 355-358; Saito et al., 1992, Assis. Reprod. Tech. Andro. 259:257-266; and Terlouw et al., 1992, Theriogenology 37: 309, each of whichis incorporated herein by reference in its entirety including allfigures, tables and drawings. Nuclear transfer may be accomplished byusing oocytes that are not surrounded by a zona pellucida.

The term “cryopreserving” as used herein can refer to freezing a cell,embryo, or animal of the invention. Cells, embryos, or portions ofanimals of the invention are frozen at temperatures preferably lowerthan 0° C., more preferably lower than −80° C., and most preferably attemperatures lower than −196° C. Cells and embryos of the invention canbe cryopreserved for an indefinite amount of time. It is known thatbiological materials can be cryopreserved for more than fifty years andstill remain viable. For example, bovine semen that is cryopreserved formore than fifty years can be utilized to artificially inseminate afemale bovine animal and result in the birth of a live offspring.Methods and tools for cryopreservation are well-known to those skilledin the art. See, e.g., U.S. Pat. No. 5,160,312, entitled“Cryopreservation Process for Direct Transfer of Embryos,” issued toVoelkel on Nov. 3, 1992.

The term “thawing” as used herein can refer to a process of increasingthe temperature of a cryopreserved cell, embryo, or portions of animals.Methods of thawing cryopreserved materials such that they are activeafter a thawing process are well-known to those of ordinary skill in theart.

The terms “transfected” and “transfection” as used herein refer tomethods of delivering exogenous DNA into a cell. These methods involve avariety of techniques, such as treating cells with high concentrationsof salt, an electric field, liposomes, polycationic micelles, ordetergent, to render a host cell outer membrane or wall permeable tonucleic acid molecules of interest. These specified methods are notlimiting and the invention relates to any transformation technique wellknown to a person of ordinary skill in the art. See, e.g., MolecularCloning, a Laboratory Manual, 2nd Ed., 1989, Sambrook, Fritsch, andManiatis, Cold Spring Harbor Laboratory Press and Transgenic Animals,Generation and Use, 1997, Edited by L. M. Houdebine, Hardwood AcademicPublishers, Australia, both of which were previously incorporated byreference.

The term “foreign DNA” as used herein can refer to DNA that can betransfected into a target cell, where foreign DNA harbors at least onebase pair modification as compared to the nuclear DNA of the targetorganism. Foreign DNA and transfection can be further understood anddefined in conjunction with the term “modified nuclear DNA,” describedpreviously.

The term “dissociating” as used herein can refer to materials andmethods useful for separating a cell away from another cell, where thecells originally contacted one another. For example, a blastomere (i.e., a cellular member of a morula stage embryo) can be pulled away fromthe rest of a developing cell mass by techniques and apparatus wellknown to a person of ordinary skill in the art. See, e.g, U.S. Pat. No.4,994,384, entitled “Multiplying Bovine Embryos,” issued on Feb. 19,1991, hereby incorporated herein by reference in its entirety, includingall figures, tables, and drawings. Alternatively, cells proliferating inculture can be separated from one another to facilitate such processesas cell passaging and formation of EG cells, which are described herein.In addition, dissociation of a cultured cell from a group of culturedcells can be useful as a first step in a process of nuclear transfer, asdescribed hereafter. When a cell is dissociated from an embryo, adissociation can be useful for such processes as re-cloning, a processdescribed herein, as well as a step for multiplying a number of embryos.

In another aspect, the invention features a totipotent porcine cell,prepared by a process comprising the steps of: (a) isolating at leastone precursor cell; and (b) culturing the precursor cell in a cellculture media; where at least one cell in the culture media develops amorphology of an embryonic germ cell. In preferred embodiments, (1) theprocess comprises the step of introducing a stimulus to the precursorcell that converts the precursor cell into the totipotent porcine cell;(2) the process comprises the step of culturing the precursor cell in acell culture medium that comprises a significant concentration of atleast one carbohydrate; (3) the carbohydrate is glucose; and (4) thecell culture medium comprises one or more antibiotics.

The term “converts” as used herein can refer to the phenomenon in whichprecursor cells become totipotent. The term “convert” is synonymous withthe term “reprogram” as used herein when the precursor cell isnon-totipotent. Precursor cells can be converted into totipotent cellsin varying proportions. For example, it is possible that only a smallportion of precursor cells are converted into totipotent cells.

The term “stimulus” as used herein can refer to materials and/or methodsuseful for converting precursor cells into totipotent cells. A stimuluscan be electrical, mechanical, temperature-related, and/or chemical, forexample. The stimulus may be a combination of one or more differenttypes of stimuli. A stimulus can be introduced to precursor cells forany period of time that accomplishes the conversion of precursor cellsinto totipotent cells.

The term “introduce” as used herein in reference to a stimulus can referto a step or steps in which precursor cells are contacted with astimulus. If a stimulus is chemical in nature, for example, such astimulus may be introduced to precursor cells by mixing the stimuluswith a cell culture medium.

The term “significant concentration of at least one carbohydrate” asused herein can refer to a cell culture medium having at least onecarbohydrate in a concentration that does not lyse or shrink culturedcells. Cultured cells can lyse or shrink when osmotic pressure of aculture media is too great. Cells may tolerate a wide range ofosmolarities (e.g., between 260 mOsm/kg and 320 mOsm/kg). Increasingconcentrations of carbohydrates in culture media can dramaticallyincrease osmotic pressure of a culture medium, which can effect cellviability. See, e.g., Cells: a laboratory manual (vol. 1), 1998, D. L.Spector, R. D. Goldman, L. A. Leinwand (eds.), Cold Spring HarborLaboratory Press, hereby incorporated herein by reference in itsentirety, including all figures, tables, and drawings.

A carbohydrate can be any monosaccharide, disaccharide, orpolysaccharide known in the art. Examples of carbohydrates include, butare not limited to, glucose, mannose, dextrose, mannose, idose,galactose, talose, gulose, altrose, allose, ribose, arabinose, xylose,lyxose, threose, erythrose, glyceraldehyde, sucrose, lactose, maltose,cellulose, and glycogen. An especially preferred carbohydrate isglucose. Preferred concentrations of glucose in cell culture media aregreater than 1 mM glucose, more greater than 5 mM glucose, 10 mMglucose, and 15 mM glucose, and most preferably about 17 mM glucose. Theterm “about” as used in relation to glucose concentrations can refer toplus or minus 2 mM glucose.

The term “antibiotic” as used herein can refer to any molecule thatdecreases growth rates of a bacterium, yeast, fungi, mold, or othercontaminants in a cell culture. Antibiotics are optional components ofcell culture media. Examples of antibiotics are well known in the art.See, Sigma and DIFCO catalogs.

In preferred embodiments (1) the precursor cells are co-cultured withfeeder cells; (2) the precursor cells are not co-cultured with feedercells; (3) the feeder cells are established from fetal cells; (4) thefetal cells arise from a fetus where no cell types have been removedfrom the fetus (e.g., the entire fetus is dissociated and placed in acell culture system); (5) the fetal cells arise from a fetus where oneor more cell types have been removed from the fetus (e.g., the headregion is removed and the remaining fetus is dissociated and placed in acell culture system); (6) a stimulus is introduced to precursor cells byfeeder cells; (7) the feeder cells are the only source of the stimulus;(8) the stimulus is introduced to the precursor cells in a mechanicalfashion; (9) the only stimulus that is introduced to the precursor cellsis introduced in a mechanical fashion; (10) the stimulus is introducedto the precursor cells by feeder cells and in a mechanical fashion; (11)the stimulus comprises the step of incubating the precursor cells with areceptor ligand cocktail; (12) the precursor cells are isolated from anungulate animal and preferably a porcine animal; (13) the precursorcells are selected from the group consisting of non-embryonic cells,non-fetal cells, differentiated cells, undifferentiated cells, somaticcells, embryonic cells, fetal cells, embryonic stem cells, primordialgerm cells, genital ridge cells, cumulus cells, amniotic cells, fetalfibroblast cells, hepatacytes, embryonic germ cells, adult cells, cellsisolated from an asynchronous population of cells, and cells isolatedfrom a synchronized population of cells where the synchronous populationis not arrested in the G₀ stage of the cell cycle; (14) the receptorligand cocktail comprises at least one component selected from the groupconsisting of cytokine, growth factor, trophic factor, and neurotrophicfactor, LIF, and FGF; (15) the LIF has an amino acid sequencesubstantially similar to the amino acid sequence of human LIF; and (16)the FGF has an amino acid sequence substantially similar to the aminoacid sequence of bovine bFGF.

The terms “mechanical fashion” and “mechanical stimulus” as used hereincan refer to introducing a stimulus to cells where the stimulus is notintroduced by feeder cells. For example, purified LIF and bFGF (definedhereafter) can be introduced as a stimulus to precursor cells by addingthese purified products to a cell culture medium in which the precursorcells are growing. Also as explained herein, a significant amount ofglucose may be added to a culture medium as a stimulus to cells.

The term “feeder cells” as used herein can refer to cells that aremaintained in culture and are co-cultured with target cells. Targetcells can be precursor cells, embryonic stem cells, embryonic germcells, cultured cells, and totipotent cells, for example. Feeder cellscan provide, for example, peptides, polypeptides, electrical signals,organic molecules (e.g., steroids), nucleic acid molecules, growthfactors (e.g., bFGF), other factors (e.g., cytokines such as LIF andsteel factor), and metabolic nutrients to target cells. Certain cells,such as embryonic germ cells, cultured cells, and totipotent cells maynot require feeder cells for healthy growth. Feeder cells preferablygrow in a mono-layer.

Feeder cells can be established from multiple cell types. Examples ofthese cell types are fetal cells, mouse cells, Buffalo rat liver cells,and oviductal cells. These examples are not meant to be limiting. Tissuesamples can be broken down to establish a feeder cell line by methodswell known in the art (e.g., by using a blender). Feeder cells mayoriginate from the same or different animal specie as precursor cells.Feeder cells can be established from ungulate fetal cells, porcine fetalcells, and murine fetal cells. One or more cell types can be removedfrom a fetus (e.g., primordial germs cells, cells in the head region,and cells in the body cavity region) and a feeder layer can beestablished from those cells that have been removed or cells in theremaining dismembered fetus. When an entire fetus is utilized toestablish fetal feeder cells, feeder cells (e.g., fibroblast cells) andprecursor cells (e.g., primordial germ cells) can arise from the samesource (e.g., one fetus).

The term “receptor ligand cocktail” as used herein can refer to amixture of one or more receptor ligands. A receptor ligand can refer toany molecule that binds to a receptor protein located on the outside orthe inside of a cell. Receptor ligands can be selected from molecules ofthe cytokine family of ligands, neurotrophin family of ligands, growthfactor family of ligands, and mitogen family of ligands, all of whichare well known to a person of ordinary skill in the art. Examples ofreceptor/ligand pairs are: epidermal growth factor receptor/epidermalgrowth factor, insulin receptor/insulin, cAMP-dependent proteinkinase/cAMP, growth hormone receptor/growth hormone, and steroidreceptor/steroid. It has been shown that certain receptors exhibitcross-reactivity. For example, heterologous receptors, such asinsulin-like growth factor receptor 1 (IGFR1) and insulin-like growthfactor receptor 2 (IGFR2) can both bind IGF1. When a receptor ligandcocktail comprises a stimulus, the receptor ligand cocktail can beintroduced to a precursor cell in a variety of manners known to a personof ordinary skill in the art.

The term “cytokine” as used herein can refer to a large family ofreceptor ligands well-known to a person of ordinary skill in the art.The cytokine family of receptor ligands includes such members asleukemia inhibitor factor (LIF); cardiotrophin 1 (CT-1); ciliaryneurotrophic factor (CNTF); stem cell factor (SCF), which is also knownas Steel factor; oncostatin M (OSM); and any member of the interleukin(IL) family, including IL-6, IL-1, and IL-12. The teachings of theinvention do not require the mechanical addition of steel factor (alsoknown as stem cell factor in the art) for the conversion of precursorcells into totipotent cells.

The term “growth factor” as used herein can refer to any receptor ligandthat may cause a cell growth effect, may cause a cell proliferationeffect, and/or may effect cell morphology. Examples of growth factorsare well known in the art. Fibroblast growth factor (FGF) is one exampleof a growth factor. The term “bFGF” can refer to basic FGF.

The term “substantially similar” as used herein in reference to aminoacid sequences can refer to two amino acid sequences having preferably50% or more amino acid identity, more preferably 70% or more amino acididentity or most preferably 90% or more amino acid identity. Amino acididentity is a property of amino acid sequence that measures theirsimilarity or relationship. Identity is measured by dividing the numberof identical residues in the two sequences by the total number ofresidues and multiplying the product by 100. Thus, two copies of exactlythe same sequence have 100% identity, while sequences that are lesshighly conserved and have deletions, additions, or replacements have alower degree of identity. Those of ordinary skill in the art willrecognize that several computer programs are available for performingsequence comparisons and determining sequence identity.

When precursor cells are cultured in vitro, it has been discovered thatprecursor cells can give rise to cells having a different cellmorphology than the precursor cells without introducing the precursorcells to a stimulus. For example, it has been discovered that precursorgenital ridge cells can develop into cells having EG cell morphologywithout contacting the precursor cells with feeder cells, a receptorligand, or a growth factor. Thus, in preferred embodiments, (1)precursor cells are not contacted with exogenous receptor ligand; (2)precursor cells are not contacted with exogenous growth factor; (3)precursor cells are not contacted with feeder cells; (4) precursor cellsare not contacted with exogenous receptor ligand and are not contactedwith exogenous growth factor; (5) precursor cells are not contacted withexogenous receptor ligand and are not contacted with feeder cells; (6)precursor cells are not contacted with exogenous growth factor and arenot contacted with feeder cells; and (7) precursor cells are notcontacted with exogenous receptor ligand and are not contacted withexogenous growth factor and are not contacted with feeder cells.

The term “exogenous” as used herein in reference to growth factor orreceptor ligand can refer to an outside source of a receptor ligandand/or growth factor that may be added to a substrate or medium that isin contact with target cells. For example, purified bFGF that iscommercially available to a person of ordinary skill in the art may beadded to cell culture media that contacts precursor cells. In thislatter example, such purified bFGF can be referred to as “exogenousbFGF.” Multiple exogenous receptor ligands and/or multiple exogenousgrowth factors or combinations thereof may be added to a liquid mediumcontacting cells. Alternatively, it may not be required that precursorcells are contacted with exogenous growth factor or exogenous receptorligand, as discussed previously.

In another aspect, the invention features a method for preparing atotipotent porcine cell, comprising the following steps: (a) isolatingone or more precursor cells; and (b) introducing the precursor cell to astimulus that converts the precursor cell into the totipotent cell. Anyof the embodiments defined previously herein in reference to totipotentporcine cells relate to methods for preparing totipotent porcine cells.In yet another aspect, the invention features a method for preparing atotipotent porcine cell, comprising the following steps: (a) isolatingat least one precursor cell; and (b) culturing the precursor cell in acell culture media to establish the totipotent cell; where thetotipotent cell has a morphology of an embryonic germ cell.

Cloned Embrvos of the Invention

The invention relates in part to a cloned totipotent porcine embryo.Hence, aspects of the invention feature a cloned porcine embryo where(1) the embryo is totipotent; (2) the embryo arises from a totipotentcell; (3) the embryo arises from a non-embryonic porcine cell; and (4)any combination of the foregoing.

The term “totipotent” as used herein in reference to embryos can referto embryos that can develop into a live born porcine animal. The term“live born” is defined previously.

The term “cloned” as used herein can refer to a cell, embryonic cell,fetal cell, and/or animal cell having a nuclear DNA sequence that issubstantially similar or identical to a nuclear DNA sequence of anothercell, embryonic cell, fetal cell, and/or animal cell. The terms“substantially similar” and “identical” are described herein. A clonedembryo can arise from one nuclear transfer process, or alternatively, acloned embryo can arise from a cloning process that includes at leastone re-cloning step. If a cloned embryo arises from a cloning procedurethat includes at least one re-cloning step, then the cloned embryo canindirectly arise from a totipotent cell since the re-cloning step canutilize embryonic cells isolated from an embryo that arose from atotipotent cell.

In preferred embodiments (1) the cloned porcine embryo can be one memberof a plurality of embryos, where the plurality of embryos share asubstantially similar nuclear DNA sequence; (2) the cloned porcineembryo can be one member of a plurality of embryos, and the plurality ofembryos can have an identical nuclear DNA sequence; (3) the clonedporcine embryo has a nuclear DNA sequence that is substantially similarto a nuclear DNA sequence of a live born porcine animal; (4) one or morecells of the cloned porcine embryo have modified nuclear DNA; (5) thecloned porcine embryo is subject to manipulation; (6) the manipulationcomprises the step of culturing the embryo in a suitable medium; (7) themedium can comprise feeder cells; (8) the manipulation of an embryocomprises the step of implanting the embryo into reproductive tract of afemale animal; (9) the female animal is preferably an ungulate animaland more preferably a porcine animal; (10) the estrus cycle of thefemale is synchronized with the development cycle of the embryo; and(11) the manipulation comprises the step of incubating the embryo in anartificial environment.

All preferred embodiments related to modified nuclear DNA for totipotentcells of the invention extend to cloned embryos of the invention. Inaddition, any of the manipulations described in conjunction withtotipotent cells of the invention apply to cloned embryos of theinvention.

The term “substantially similar” as used herein in reference to nuclearDNA sequences refer to two nuclear DNA sequences that are nearlyidentical. Two sequences may differ by copy error differences thatnormally occur during replication of nuclear DNA. Substantially similarDNA sequences are preferably greater than 97% identical, more preferablygreater than 98% identical, and most preferably greater than 99%identical. The term “identity” as used herein in reference to nuclearDNA sequences can refer to usage of the term in reference to amino acidsequences, which is described previously herein. It is preferred andexpected that nuclear DNA sequences are identical for cloned animals.Examples of methods for determining whether cloned animals and cellsfrom which they are cloned have substantially similar or identicalnuclear DNA sequences are microsattelite analysis and DNA fingerprintinganalysis. Ashworth et al., 1998, Nature 394: 329 and Signer et al.,1998, Nature 394: 329.

The term “plurality” as used herein in reference to embryos can refer toa set of embryos having a substantially similar nuclear DNA sequence. Inpreferred embodiments, a plurality consists of five or more embryos, tenor more embryos, fifteen or more embryos, twenty or more embryos, fiftyor more embryos, seventyfive or more embryos, one-hundred or moreembryos, and one-thousand or more embryos.

The term “culturing” as used herein with respect to embryos can refer tolaboratory procedures that involve placing an embryo in a culturemedium. An embryo can be placed in a culture medium for an appropriateamount of time to allow stasis of an embryo, or to allow the embryo togrow in the medium. Culture media suitable for culturing embryos arewell-known to those skilled in the art. See, e.g., Nagashima et al.,1997, Mol. Reprod. Dev. 48: 339-343; Petters & Wells, 1993, J. Reprod.Fert. (Suppl) 48: 61-73; Reed et al., 1992, Theriogenology 37: 95-109;Dobrinsky et al., 1996, Biol. Reprod. 55: 1069-1074; U.S. Pat. No.5,213,979, First et al., “In Vitro Culture of Bovine Embryos,” May 25,1993; U.S. Pat. No. 5,096,822, Rosenkrans, Jr. et al., “Bovine EmbryoMedium,” Mar. 17, 1992, each of which is incorporated herein byreference in its entirety, including all figures, tables, and drawings.

The term “suitable medium” as used herein can refer to any medium thatallows cell proliferation or allows stasis of an embryo. If a mediumallows cell proliferation, a suitable medium need not promote maximumproliferation, only measurable cell proliferation. A suitable medium forembryo development can be an embryo culture medium described herein byexample. The term “feeder cells” is defined previously herein. Embryosof the invention can be cultured in media with or without feeder cells.In other preferred embodiments, the feeder cells can be cumulus cells.

The term “implanting” as used herein in reference to embryos can referto impregnating a female animal with an embryo described herein.Implanting techniques are well known to a person of ordinary skill inthe art. See, e.g., Polge & Day, 1982, “Embryo transplantation andpreservation,” Control of Pig Reproduction, DJA Cole and GR Foxcroft,eds., London, UK, Butterworths, pp. 227-291; Gordon, 1997, “Embryotransfer and associated techniques in pigs,” Controlled reproduction inpigs (Gordon, ed), CAB International, Wallingford UK, pp 164-182; andKojima, 1998, “Embryo transfer,” Manual of pig embryo transferProcedures, National Livestock Breeding Center, Japanese Society forDevelopment of Swine Technology, pp 76-79, each of which is incorporatedherein by reference in its entirety, including all figures, tables, anddrawings.

The embryo may be allowed to develop in utero, or alternatively, thefetus may be removed from the uterine environment before parturition.

The term “synchronized” as used herein in reference to estrus cycle, canrefer to assisted reproductive techniques well known to a person ofordinary skill in the art. These techniques are fully described in thereference cited in the previous paragraph. Typically, estrogen andprogesterone hormones are utilized to synchronize the estrus cycle ofthe female animal with the developmental stage of the embryo. The term“developmental stage” as used herein can refer to embryos of theinvention and morphological and biochemical changes during embryodevelopment. This developmental process is predictable for embryos fromungulates, and can be synchronized with the estrus cycle of a recipientanimal. A procedure for synchronizing a female porcine animal is setforth hereafter.

The term “artificial environment” refers to one that promotesdevelopment of an embryo or other developing cell mass. An artificialenvironment can be a uterine environment or an oviductal environment ofa species different from that of a developing cell mass. For example, adeveloping bovine embryo can be placed into an uterus or oviduct of anovine animal. Stice & Keefer, 1993, “Multiple generational bovine embryocloning,” Biology of Reproduction 48: 715-719. Alternatively, anartificial development environment can be assembled in vitro. This typeof artificial uterine environment can be synthesized using biologicaland chemical components known in the art.

In another aspect the invention features a cloned mammalian embryo,where the embryo is totipotent, prepared by a process comprising thestep of nuclear transfer. Preferably, nuclear transfer occurs between(a) a nuclear donor, and (b) an oocyte, where the oocyte is at a stageallowing formation of the embryo.

In preferred embodiments, (1) the oocyte is an enucleated oocyte; (2)the oocyte preferably originates from an ungulate animal and morepreferably originate from a porcine animal; (3) the oocyte has beenmatured; (4) the oocyte has been matured for more than 48 hours; (5) theoocyte has been matured for about 53 hours; (6) the nuclear donor isplaced in the perivitelline space of the oocyte; (7) the nuclear donorutilized for nuclear transfer can arise from any of the cells describedpreviously (e.g., a non-embryonic cell, a primordial germ cell, agenital ridge cell, a differentiated cell, a fetal cell, a non-fetalcell, a non-primordial germ cell, a cell isolated from an asynchronouspopulation of cells, a cell isolated from a synchronous population ofcells, a cell isolated from an existing animal, an embryonic stem cell,an embryonic germ cell, an amniotic cell, a cumulus cell, and a fetalfibroblast cell); (8) the nuclear transfer comprises the step oftranslocation of the nuclear donor into the recipient oocyte; (9) thetranslocation can comprise the step of injection of the nuclear donorinto the recipient oocyte; (10) the translocation can comprise the stepof fusion of the nuclear donor and the oocyte; (11) the fusion cancomprise the step of delivering one or more electrical pulses to thenuclear donor and the oocyte; (12) the fusion can comprise the step ofdelivering a suitable concentration of at least one fusion agent to thenuclear donor and the oocyte; (13) the nuclear transfer may comprise thestep of activation of the nuclear donor and the oocyte; (14) theactivation is accomplished by (i) increasing intracellular levels ofdivalent cations in a cell, and (ii) reducing phosphorylation ofcellular proteins in the cell; (15) the activation is accomplished by(i) introducing a divalent ion ionophore to a cell, and (ii) introducinga protein kinase inhibitor to a cell; (16) the divalent ion ionophore isa Ca²⁺ ionophore; (17) the Ca²⁺ ionophore is ionomycin; (18) the proteinkinase inhibitor is DMAP; and (19) activation is accomplished byintroducing DMAP and ionomycin to a cell.

The term “nuclear donor” as used herein can refer to a cell or a nucleusfrom a cell that is translocated into a nuclear acceptor. A nucleardonor may be a totipotent porcine cell. In addition, a nuclear donor maybe any cell described herein, including, but not limited to anon-embryonic cell, a non-fetal cell, a differentiated cell, a somaticcell, an embryonic cell, a fetal cell, an embryonic stem cell, aprimordial germ cell, a genital ridge cell, a cumulus cell, an amnioticcell, a fetal fibroblast cell, a hepatacyte, an embryonic germ cell, anadult cell, a cell isolated from an asynchronous population of cells,and a cell isolated from a synchronized population of cells where thesynchronous population is not arrested in the G₀ stage of the cellcycle. A nuclear donor cell can also be a cell that has differentiatedfrom an embryonic stem cell. See, e.g., Piedrahita et al., 1998, Biol.Reprod 58: 1321-1329; Shim et al., 1997, Biol. Reprod. 57: 1089-1095;Tsung et al., 1995, Shih Yen Sheng Wu Hsueh Pao 28: 173-189; andWheeler, 1994, Reprod Fertil. Dev. 6: 563-568, each of which isincorporated herein by reference in its entirety including all figures,drawings, and tables. In addition, a nuclear donor may be a cell thatwas previously frozen or cryopreserved.

The term “enucleated oocyte” as used herein can refer to an oocyte whichhas had its nucleus removed. Typically, a needle can be placed into anoocyte and the nucleus can be aspirated into the needle. The needle canbe removed from the oocyte without rupturing the plasma membrane. Thisenucleation technique is well known to a person of ordinary skill in theart. See, U.S. Pat. No. 4,994,384; U.S. Pat. No. 5,057,420; andWilladsen, 1986, Nature 320:63-65. An enucleated oocyte is preferablyprepared from an oocyte that has been matured for greater than 24 hours,preferably matured for greater than 36 yours, more preferably maturedfor greater than 48 hours, and most preferably matured for about 53hours.

The terms “maturation” and “matured” as used herein can refer to aprocess in which an oocyte is incubated in a medium in vitro. Maturationmedia can contain multiple types of components, including hormones andgrowth factors. Time of maturation can be determined from the time thatan oocyte is placed in a maturation medium to the time that the oocyteis subject to a manipulation (e.g., enucleation, nuclear transfer,fusion, and/or activation). Oocytes can be matured in multiple mediawell known to a person of ordinary skill in the art. See, e.g., Mattioliet al., 1989, Theriogenology 31: 1201-1207; Jolliff & Prather, 1997,Biol. Reprod. 56: 544-548; Funahashi & Day, 1993, J. Reprod. Fert. 98:179-185; Nagashima et al., 1997, Mol. Reprod. Dev. 38: 339-343;Abeydeera et al., 1998, Biol. Reprod. 58: 213-218; Funahashi et al.,1997, Biol. Reprod. 57: 49-53; and Sawai et al., 1997, Biol. Reprod. 57:1-6, each of which is incorporated herein by reference in its entirety,including all figures, tables, and drawings. Oocytes can be matured forany period of time: an oocyte can be matured for greater than 10 hours,greater than 20 hours, greater than 24 hours, greater than 60 hours,greater than 72 hours, greater than 90 hours, preferably matured forgreater than 36 hours, more preferably matured for greater than 48hours, and most preferably matured for about 53 hours. The term “about”with respect to oocyte maturation can refer to plus or minus 3 hours.

An oocyte can also be matured in vivo. Time of maturation may be thetime that an oocyte receives an appropriate stimulus to resume meiosisto the time that the oocyte is manipulated. Similar maturation periodsdescribed above for in vitro matured oocytes apply to in vivo maturedoocytes.

A variety of oocytes can be selected for maturation. For example,oocytes can be isolated from a pre-pubertal porcine animal or aperi-pubertal animal (e.g., a sow). However, oocytes from pre-pubertalporcine animals may be incapable of spontaneous resumption of meiosis invitro. It is a preferred embodiment of the invention that oocytesisolated from a sow are utilized for maturation and eventually innuclear transfer procedures.

Nuclear transfer may be accomplished by combining one nuclear donor andmore than one enucleated oocyte. In addition, nuclear transfer may beaccomplished by combining one nuclear donor, one or more enucleatedoocytes, and the cytoplasm of one or more enucleated oocytes.

The term “cybrid” as used herein can refer to an oocyte having a nucleardonor inserted within. The term “cybrid” can refer to an oocyte having anuclear donor that is translocated into the oocyte. A nuclear donor maybe fused with an oocyte, and the term “cybrid” includes oocytes that arenot fused with a nuclear donor.

The invention relates in part to cloned mammalian embryos established bynuclear transfer of a nuclear donor and an non-enucleated oocyte. Acloned embryo may be established where nuclear DNA from the donor cellreplicates during cellular divisions while nuclear DNA from an oocytedoes not replicate. See, e.g., Wagoner et al., 1996, “Functionalenucleation of bovine oocytes: effects of centrifugation and ultravioletlight,” Theriogenology 46: 279-284.

The term “another ungulate” as used herein can refer to a situationwhere a nuclear donor originates from an ungulate of a differentspecies, genera or family than the ungulate from which the recipientoocyte originates. For example, a porcine cell can be used as a nucleardonor, while a recipient oocyte can be isolated from a domestic cow.This example is not meant to be limiting and any ungulate species/familycombination of nuclear donors and recipient oocytes are foreseen by theinvention.

The term “translocation” as used herein in reference to nuclear transfercan refer to combining a nuclear donor and a recipient oocyte.Translocation may be performed by such techniques as fusion and/ordirect injection, for example.

The term “injection” as used herein in reference to embryos, can referto perforation of an oocyte with a needle, and insertion of a nucleardonor in the needle into the oocyte. In preferred embodiments, a nucleardonor may be injected into the cytoplasm of an oocyte or in theperivitelline space of an oocyte. This direct injection approach is wellknown to a person of ordinary skill in the art, as indicated bypublications already incorporated herein in reference to nucleartransfer. For a direct injection approach to nuclear transfer, a wholecell may be injected into an oocyte, or alternatively, a nucleusisolated from a cell may be injected into an oocyte. Such an isolatednucleus may be surrounded by nuclear membrane only, or the isolatednucleus may be surrounded by nuclear membrane and plasma membrane in anyproportion. An oocyte may be pre-treated to enhance the strength of itsplasma membrane, such as by incubating the oocyte in sucrose prior toinjection of a nuclear donor.

Techniques for placing a nuclear donor into the perivitelline space ofan enucleated oocyte is well known to a person of ordinary skill in theart, and is fully described in patents and references cited previouslyherein in reference to nuclear transfer.

The term “fusion” as used herein can refer to combination of portions oflipid membranes corresponding to a nuclear donor and a recipient oocyte.Lipid membranes can correspond to plasma membranes of cells or nuclearmembranes, for example. Fusion can occur with addition of a fusionstimulus between a nuclear donor and recipient oocyte when they areplaced adjacent to one another, or when a nuclear donor is placed in theperivitelline space of a recipient oocyte, for example. Specificexamples for translocation of a porcine mammalian cell into an oocyteare described hereafter in other preferred embodiments. These techniquesfor translocation are fully described in references cited previouslyherein in reference to nuclear transfer.

The term “electrical pulses” as used herein can refer to subjecting anuclear donor and recipient oocyte to electric current. For nucleartransfer, a nuclear donor and recipient oocyte can be aligned betweenelectrodes and subjected to electrical current. Electrical current canbe alternating current or direct current. Electrical current can bedelivered to cells for a variety of different times as one pulse or asmultiple pulses. Cells are typically cultured in a suitable medium fordelivery of electrical pulses. Examples of electrical pulse conditionsutilized for nuclear transfer are described in references and patentspreviously cited herein in reference to nuclear transfer.

The term “fusion agent” as used herein can refer to any compound orbiological organism that can increase the probability that portions ofplasma membranes from different cells will fuse when a nuclear donor isplaced adjacent to a recipient oocyte. In preferred embodiments fusionagents are selected from the group consisting of polyethylene glycol(PEG), trypsin, dimethylsulfoxide (DMSO), lectins, agglutinin, viruses,and Sendai virus. These examples are not meant to be limiting and otherfusion agents known in the art are applicable and included herein.

The term “suitable concentration” as used herein in reference to fusionagents, can refer to any concentration of a fusion agent that affords ameasurable amount of fusion. Fusion can be measured by multipletechniques well known to a person of ordinary skill in the art, such asby utilizing a light microscope, dyes, and fluorescent lipids, forexample.

The term “activation” can refer to any materials and methods useful forstimulating a cell to divide before, during, and after a nucleartransfer step. The term “cell” as used in the previous sentence canrefer to an oocyte, a cybrid, a nuclear donor, and an early stageembryo. These types of cells may require stimulation in order to divideafter nuclear transfer has occurred. The invention pertains to anyactivation materials and methods known to a person of ordinary skill inthe art.

Although electrical pulses are sometimes sufficient for stimulatingactivation of cells, other non-electrical means for activation areuseful and are often necessary for proper activation of a cell. Chemicalmaterials and methods useful for nonelectrical activation are describedbelow in other preferred embodiments of the invention. When two or morechemical components are introduced to a cell for activating the cell,the components can be added simultaneously or in steps.

Examples of electrical processes for activation are well known in theart. Researchers have also reported non-electrical processes foractivation. See, e.g., Grocholová et al., 1997, J. Exp. Zoology 277:49-56; Schoenbeck et al., 1993, Theriogenology 40: 257-266; Prather etal., 1989, Biology of Reproduction 41: 414-418; Prather et al., 1991,Molecular Reproduction and Development 28: 405-409; Mattioli et al.,1991, Molecular Reproduction and Development 30: 109-125; Terlouw etal., 1992, Theriogenology 37: 309; Prochazka et al., 1992, J. Reprod.Fert. 96: 725-734; Funahashi et al., 1993, Molecular Reproduction andDevelopment 36: 361-367; Prather et al., Bio. Rep. Vol. 50 Sup 1: 282;Nussbaum et al., 1995, Molecular Reproduction and Development 41: 70-75;Funahashi et al., 1995, Zygote 3: 273-281; Wang et al., 1997, Biology ofReproduction 56: 1376-1382; Piedrahita et al., 1989, Biology ofReproduction 58: 1321-1329; Macháty et al., 1997, Biology ofReproduction 57: 85-91; and Macháty et al., 1995, Biology ofReproduction 52: 753-758.

Examples of components that are useful for non-electrical activationinclude ethanol; inositol trisphosphate (IP₃); divalent ions (e.g.,addition of Ca²⁺ and/or Sr²⁺); microtubule inhibitors (e.g.,cytochalasin B); ionophores for divalent ions (e.g., the Ca³⁺ ionophoreionomycin); protein kinase inhibitors (e.g., 6-dimethylaminopurine(DMAP)); protein synthesis inhibitors (e.g., cyclohexamide); phorbolesters such as phorbol 12-myristate 13-acetate (PMA); and thapsigargin.It is also known that temperature change and mechanical techniques arealso useful for non-electrical activation. The invention includes anyactivation techniques known in the art. See, e.g., U.S. Pat. No.5,496,720, entitled “Parthenogenic Oocyte Activation,” issued on Mar. 5,1996, Susko-Parrish et al., and Wakayama et al., 1998, Nature 394:369-374, each of which is incorporated herein by reference in itsentirety, including all figures, tables, and drawings.

When ionomycin and DMAP are utilized for non-electrical activation,ionomycin and DMAP may be introduced to cells simultaneously or in astep-wise addition, the latter being a preferred mode as describedherein. Preferred concentrations of ionomycin and DMAP are 0.5 μMionomycin to 50 μM ionomycin and 0.5 mM DMAP to 50 mM DMAP, morepreferably 1 μM ionomycin to 20 μM ionomycin and 1 mM DMAP to 5 mM DMAP,and most preferably about 10 μM ionomycin and about 2 mM DMAP, where theterm “about” can refer to plus or minus 2 μM ionomycin and 1 mM DMAP.

In other preferred embodiments, (1) one or more cells of the clonedporcine embryo comprise modified nuclear DNA; (2) the cloned porcineembryo is subject to manipulation; (3) the manipulation comprises thestep of disaggregating at least one individual cell from a clonedembryo; (4) the manipulation comprises the step of utilizing theindividual cell as a nuclear donor in a nuclear transfer procedure; (5)the individual cell is disaggregated from the inner cell mass of ablastocyst stage embryo; (6) the individual cell is disaggregated from apre-blastocyst stage embryo; (7) the manipulation comprises the processof re-cloning; (8) the re-cloning process comprises the steps of: (a)separating the embryo into one or more individual cells, and (b)performing at least one subsequent nuclear transfer between (i) anindividual cell of (a), and (ii) an oocyte; (9) the individual cell isplaced in the perivitelline space of the enucleated oocyte for thesubsequent nuclear transfer; (10) the subsequent nuclear transfercomprises at least one of the steps of translocation, injection, fusion,and activation of the individual cell and/or the enucleated oocyte; (11)one or more cells of the cloned mammalian embryo arising from thesubsequent nuclear transfer comprises modified nuclear DNA; and (12) thecloned mammalian embryo arising from the subsequent nuclear transfer maybe subject to a subsequent manipulation, where the subsequentmanipulation is any of the manipulation steps defined previously hereinin relation to totipotent cells and/or cloned embryos.

The term “individual cells” as used herein can refer to cells that havebeen isolated from a cloned mammalian embryo of the invention. Anindividual single cell can be isolated from an embryo by techniques wellknown to those skilled in the art, as discussed in references citedpreviously herein.

The term “subsequent nuclear transfer” as described herein is alsoreferred to as a “re-cloning” step. Preferably, a re-cloning step can beutilized to enhance nuclear reprogramming during nuclear transfer, suchthat a product of nuclear transfer is a live born animal. The number ofsubsequent nuclear transfer steps is discussed in greater detailhereafter.

Any of the preferred embodiments related to the translocation,injection, fusion, and activation steps described previously herein canrelate to any subsequent nuclear transfer step.

The term “inner cell mass” as used herein can refer to cells that giverise to the embryo proper. Cells that line the outside of a blastocystcan be referred to as a trophoblast of the embryo. Methods for isolatinginner cell mass cells from an embryo are well known to a person ofordinary skill in the art, as discussed previously. The term“pre-blastocyst” is well known in the art and is referred to previously.

The term “ovulated in vivo” as used herein can refer to an oocyte thatis isolated from an animal a certain number of hours after the animalexhibits characteristics that is associated with estrus or followinginjection of exogenous gonadatrophins known to induce ovulation. Thecharacteristics of an animal in estrus are well known to a person ofordinary skill in the art, as described in references disclosed herein.See, e.g., Gordon, 1977, “Embryo transfer and associated techniques inpigs (Gordon, ed.),” CAB International, Wallingford UK, pp. 60-76 andKojima, 1998, “Embryo transfer,” Manual of pig embryo transferprocedures, National Livestock Breeding Center, Japanese Society forDevelopment of Swine Technology, pp. 7-21, each of which is incorporatedherein by reference in its entirety including all figures, tables, anddrawings.

In another aspect the invention relates to a cloned porcine embryoproduced by a process comprising the steps of (a) translocation of anuclear donor into an oocyte to establish a nuclear transfer oocyte; and(b) non-electrical activation of the nuclear transfer oocyte toestablish the porcine embryo.

In preferred embodiments, (1) the nuclear donor is a cultured cell andis selected from any of the cell types described herein; (2) the nucleardonor is a totipotent cell or is isolated from a totipotent cell; (3)the nuclear donor is any cell type discussed herein (e.g., embryonicgerm cell, cumulus cell, amniotic cell, fibroblast cell); (4) thetranslocation comprises the step of fusion; and (5) the processcomprises the step of culturing the embryo in vitro. Any other preferredembodiments discussed herein with respect to porcine embryos, andespecially with regard to activation, pertains to this aspect of theinvention.

In another aspect the invention relates to a method for preparing acloned porcine embryo. The method comprises the step of a nucleartransfer between: (a) a nuclear donor, where the nuclear donor is atotipotent porcine cell; and (b) an oocyte, where the oocyte is at astage allowing formation of the embryo. In yet another aspect theinvention relates to a method for cloning a porcine embryo, comprisingthe steps of (a) translocation of a nuclear donor into an oocyte toestablish a nuclear transfer oocyte; and (b) non-electrical activationof the nuclear transfer oocyte to establish the porcine embryo. Inpreferred embodiments, any of the embodiments of the inventionconcerning cloned porcine embryos apply to methods for preparing clonedporcine embryos.

Cloned Fetuses of the Invention

In another aspect, the invention features a cloned porcine fetus arisingfrom a totipotent embryo of the invention. A fetus may be isolated froman uterus of a pregnant female animal and may be isolated from anotherpart of a pregnant female animal in the case of an ectopic pregnancy.

In preferred embodiments, (1) one or more cells of the fetus harbormodified nuclear DNA (defined previously herein); and (2) the fetus maybe subjected to any of the manipulations defined herein. For example,one manipulation may comprise the steps of isolating a fetus from theuterus of a pregnant female animal, isolating a cell from the fetus(e.g., a primordial germ cell), and utilizing the isolated cell as anuclear donor for nuclear transfer.

Other aspects of the invention feature (1) a cloned porcine fetusprepared by a process comprising the steps of (a) preparation of acloned porcine embryo defined previously, and (b) manipulation of thecloned porcine embryo such that it develops into a fetus; (2) a methodfor preparing a cloned porcine fetus comprising the steps of (a)preparation of a cloned porcine embryo defined previously, and (b)manipulation of the cloned porcine embryo such that it develops into afetus; (3) a method of using a cloned fetus of the invention comprisingthe step of isolating at least one cell type from a fetus (e.g., forestablishing a feeder cell layer); and (4) a method of using a clonedfetus of the invention comprising the step of separating at least onepart of a fetus into individual cells (e.g., for establishing a feedercell layer).

Cloned Porcine Animals of the Invention

In another aspect the invention features a cloned porcine animal arisingfrom a totipotent porcine cell of the invention. A cloned porcine animalcan develop from a cloned embryo that is established by a nucleartransfer process between a totipotent porcine cell and an oocyte. Atotipotent porcine cell is preferably established by utilizing any ofthe materials and methods described previously herein.

In yet another aspect the invention relates to a cloned porcine animal,where the animal is one member of a plurality of porcine animals, andwhere the plurality of animals have a substantially similar nuclear DNAsequence. The term “substantially similar” in relation to nuclear DNAsequences is defined previously herein.

In preferred embodiments, (1) the plurality consists of five or moreanimals, ten or more animals, one-hundred or more animals, andone-thousand or more animals; and (2) the plurality of animals can havean identical nuclear DNA sequence. The term “identical” in reference tonuclear DNA sequences is described previously herein.

In another aspect, the invention relates to a cloned porcine animalhaving one or more cells that comprise modified nuclear DNA. All of thepreferred embodiments relating to modified nuclear DNA describedpreviously apply to cloned porcine animals of the invention.

In yet another aspect, the invention features a method of using a clonedporcine animal, comprising the step of isolating at least one componentfrom the porcine animal.

The term “component” as used herein can relate to any portion of aporcine animal. A component can be selected from the group consisting offluid, biological fluid, cell, tissue, organ, gamete, embryo, and fetus.For example, precursor cells, as defined previously, may arise fromfluids, biological fluids, cells, tissues, organs, gametes, embryos, andfetuses isolated from cloned organisms of the invention.

The term “gamete” as used herein can refer to any cell participating,directly or indirectly, to the reproductive system of an animal. Agamete can be a specialized product from the gonads of an organism,where the gamete may transfer genetic material while participating infertilization. Examples of gametes are spermatocytes, spermatogonia,oocytes, and oogonia. Gametes can be present in fluids, tissues, andorgans collected from animals (e.g., sperm is present in semen). Theinvention relates to collection of any type of gamete from an animal.For example, methods of collecting semen and oocytes are known to aperson of ordinary skill in the art. See, e.g., Gordon, 1997,“Introduction to controlled breeding in pigs, Embryo transfer andassociated techniques in pigs,” Controlled reproduction in pigs (Gordon,ed.), CAB International, Wallingford UK, pp. 1-59; Mattioli et al.,1989, Theriogenology 31: 1207-1207; Funahashi & Day, 1993, J. Reprod.Fert. 98 179-185; Funahashi et al., 1997, Biol Reprod. 57: 49-53;Abeydeera et al., 1998, Biol. Reprod. 58: 213-218; and Sawai et al.,1997, Biol. Reprod. 57: 1-6, each of which is incorporated herein byreference in its entirety including all figures, tables, and drawings.

The term “tissue” is defined previously. The term “organ” relates to anyorgan isolated from an animal or any portion of an organ. Examples oforgans and tissues are neuronal tissue, brain tissue, spleen, heart,lung, gallbladder, pancreas, testis, ovary and kidney. These examplesare not limiting and the invention relates to any organ and any tissueisolated from a cloned animal of the invention.

In a preferred embodiments, the invention relates to (1) fluids,biological fluids, cells, tissues, organs, gametes, embryos, and fetusescan be subject to manipulation; (2) the manipulation can comprise thestep of cryopreserving the gametes, embryos, and/or fetal tissues; (3)the manipulation can comprise the step of thawing the cryopreserveditems; (4) the manipulation can comprise the step of separating thesemen into X-chromosome bearing semen and Y-chromosome bearing semen;(5) the manipulation comprises methods of preparing the semen forartificial insemination; (6) the manipulation comprises the step ofpurification of a desired polypeptide(s) from the biological fluid ortissue; (7) the manipulation comprises concentration of the biologicalfluids or tissues; (8) the manipulation can comprise the step oftransferring one or more fluids, cloned cells, cloned tissues, clonedorgans, and/or portions of cloned organs to a recipient organism (e.g.,the recipient organism may be of a different specie than the donorsource); (9) the recipient organism is non-human; and (10) the recipientorganism is human.

The term “separating” as used herein in reference to separating semencan refer to methods well known to a person skilled in the art forfractionating a semen sample into sex-specific fractions. This type ofseparation can be accomplished by using flow cytometers that arecommercially available. Methods of utilizing flow cytometers fromseparating sperm by genetic content are well known in the art. Inaddition, semen can be separated by its sex-associated characteristicsby other methods well known to a person of ordinary skill in the art.See, U.S. Pat. Nos. 5,439,362, 5,346,990, and 5,021,244, entitled“Sex-Associated Membrane Proteins and Methods for Increasing theProbability that Offspring Will Be of a Desired Sex,” Spaulding, issuedon Aug. 8, 1995, Sep. 13, 1994, and Jun. 4, 1991respectively, each ofwhich is incorporated herein by reference in its entirety including allfigures, tables, and drawings.

The term “purification” as used herein can refer to increasing thespecific activity of a particular polypeptide or polypeptides in asample. Specific activity can be expressed as a ratio between theactivity or amount of a target polypeptide and the concentration oftotal polypeptide in the sample. Activity can be catalytic activityand/or binding activity, for example. Also, specific activity can beexpressed as a ratio between the concentration of target polypeptide andthe concentration of total polypeptide. Purification methods includedialysis, centrifugation, and column chromatography techniques, whichare well-known procedures to a person of ordinary skill in the art. See,e.g., Young et al., 1997, “Production of biopharmaceutical proteins inthe milk of transgenic dairy animals,” BioPharm 10(6): 34-38.

The term “transferring” as used herein can relate to shifting a group ofcells, tissues, organs, and/or portions of organs to an animal. Cells,tissues, organs, and/or portions of organs can be, for example, (a)developed in vitro and then transferred to an animal, (b) removed from acloned porcine animal and transferred to another animal of a differentspecie, (c) removed from a cloned porcine animal and transferred toanother animal of the same specie, (d) removed from one portion of ananimal (e.g., cells from a leg of an animal) and then transferred toanother portion of the same animal (e.g., a brain of the same animal),and/or (e) any combination of the foregoing.

The term “transferring” as used herein can refer to adding fluids,cells, tissues, and/or organs to an animal and can refer to removingcells, tissues, and/or organs from an animal and replacing them withcells, tissues, and/or organs from another source. For example, neuronaltissue from a cloned porcine organism can be grafted into an appropriatearea in the nervous system of a human to treat neurological diseases(e.g., Alzheimer's disease). In another example, a heart or part of aheart may be removed from a cloned porcine animal and can be surgicallyinserted into a human from which a heart or part of the heart waspreviously removed. Surgical methods for accomplishing this preferredaspect of the invention are known to a person of ordinary skill in thesurgical arts. Transferring procedures may include the step of removingcells, tissues, fluids and/or organs from a recipient organism before atransfer step.

In other aspects the invention features (1) a cloned porcine animalprepared by a process comprising the steps of: (a) preparation of acloned porcine embryo by any one of the methods described herein forproducing such a cloned porcine embryo, and (b) manipulation of thecloned porcine embryo such that it develops into a live born animal; and(2) a process for cloning a porcine animal, comprising the steps of: (a)preparation of a cloned porcine embryo by any one of the methodsdescribed herein for preparing such a cloned porcine embryo, and (b)manipulation of the cloned mammalian embryo such that it develops into alive born porcine animal.

In preferred embodiments (1) the manipulation can comprise the step ofimplanting the embryo into a uterus of an animal; (2) the estrus cycleof the animal can be synchronized to the developmental stage of theembryo; and (3) the manipulation can comprise the step of implanting theembryo into an artificial environment.

In another aspect the invention features a process for cloning a porcineanimal, comprising the steps of (a) translocation of a nuclear donorinto an oocyte to establish a nuclear transfer oocyte; (b)non-electrical activation of the nuclear transfer oocyte to establish acloned porcine embryo; and (c) transferring the porcine embryo into arecipient female, where the porcine embryo develops into a clonedporcine animal.

In preferred embodiments, (1) the nuclear donor is a cultured cell andis selected from any of the cell types described herein; (2) the nucleardonor is a totipotent cell; (3) translocation comprises fusion; and (4)the method comprises the step of culturing the porcine embryo in vitro.Any other preferred embodiments discussed herein with respect to porcineembryos, and especially with regard to activation, pertains to thisaspect of the invention.

The summary of the invention described above is not limiting and otherfeatures and advantages of the invention will be apparent from thefollowing detailed description of the preferred embodiments, as well asfrom the claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates multiple embodiments of the invention relating to thegeneration of totipotent cells from precursor cells. The figureindicates that totipotent cells can arise from embryonic stem cells,primordial germ cells, and cells isolated from an animal. The precursorcell sources illustrated by FIG. 1 are not limiting and other precursorcell sources are described herein.

FIG. 2 illustrates multiple embodiments of the invention related topathways for establishing totipotent cell lines and cloned animals.Fibroblast cells can be isolated from any source described herein.

FIG. 3 illustrates multiple embodiments of the invention forestablishing cloned transgenic cell lines and cloned transgenic animals.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to cloning technologies related to porcineanimals. The present invention provides multiple advantages over toolsand methods currently utilized in the field of cloning porcine animals.For example, the invention relates in part to totipotent cells usefulfor cloning porcine animals. These totipotent cells can give rise tomethods of producing cloned porcine animals by utilizing virtually anytype of cell. For example, cells isolated from a live born animal can bereprogrammed into totipotent cells. This feature of the inventionprovides an ability to assess a phenotype of an existing porcine animaland then readily establish a totipotent cell line for cloning thatanimal.

In addition, totipotent cells of the invention allow for establishingcell lines from virtually any type of cell. This reprogramming method isdescribed previously herein. These totipotent cell lines offer a nearlyunlimited source of donor cells for nuclear transfer cloning techniques.Moreover, this feature provides the advantage of enhancing cloningefficiency due to the lower differentiation rates of these cell linesthan existing cell lines used for cloning. For example, embryonic stemcell lines can harbor multiple colonies of cells that are nottotipotent. Cell lines of the invention can harbor a high percentage oftotipotent cells.

Moreover, methods and processes for establishing totipotent cells,totipotent cloned embryos, and cloned animals of the invention enhancecloning efficiency. This enhanced efficiency satisfies a long felt needin the art.

I. Totipotent Porcine Cells

A. Establishing Totipotent Cells

Totipotent cells of the invention can be produced from virtually anytype of precursor cell. Preferred embodiments of the invention relate tothe following types of precursor cells: (1) embryos arising from theunion of two gametes in vitro or in vivo; (2) embryonic stem cells (EScells) arising from cultured embryonic cells (e.g., pre-blastocyst cellsand inner cell mass cells); (3) cultured and non-cultured inner cellmass cells isolated from of embryos; (4) cultured and non-culturedpre-blastocyst cells; (5) cultured and non-cultured fetal cells; (6)cultured and non-cultured primordial germ cells; (7) cultured embryonicgerm cells (EG cells) as they are defined herein; (8) cultured andnon-cultured cells isolated from an animal; (9) cultured andnon-cultured cumulus cells; (10) cultured and non-cultured amnioticcells; (11) cultured and non-cultured fetal fibroblast cells; (12)cultured and non-cultured genital ridge cells; (13) cultured andnon-cultured differentiated cells; and (14) cultured and non-culturednon-differentiated cells or undifferentiated cells.

Totipotent cells of the invention are preferably generated fromprecursor cells specified herein. Cells derived from a porcine animalcan be isolated from nearly any type of tissue. For example, anear-punch can be taken from a porcine animal, cells from the sample canbe dissociated, and dissociated cells can be subsequently cultured invitro by using cell culture techniques well known to a person ofordinary skill in the art. Examples of materials and methods forculturing primary culture cells into totipotent cells are described inexemplary embodiments hereafter.

A variety of methods for culturing cells exist in the art. See, e.g.,Culture of animal cells: a manual of basic technique (2nd. edition),Freshney, copyright 1987, Alan R. Liss, Inc., New York. Particularly,cells that are precursor cells for totipotent cells, as well astotipotent cells themselves, can be grown on feeder layers. Examples offeeder layers are well known to a person of ordinary skill in the art,and can arise from a number of different cells that are cultured invitro. See, e.g., exemplary embodiment described hereafter andStrelchenko, 1996, Theriogenology 45: 130-141; Piedrahita et al., 1990,Theriogenology 34: 879-901; Piedrahita et al., 1998, Biol. Reprod. 58:1321-1329; and Shim et al., 1997, Theriogenology 47: 245, each of whichis incorporated herein by reference in its entirety including allfigures, tables, and drawings. However, precursor cells for totipotentcells as well as totipotent cells themselves need not be grown on feederlayers.

A preferred culturing condition for these precursor cells is a cellculture medium that contains a significant amount of glucose, in anamount specified herein. The cell culture condition may contain acarbohydrate that differs from glucose and may also contain multipletypes of carbohydrates and complex carbohydrates. A wide variety ofcarbohydrates are well known to a person of ordinary skill in the art.See, e.g., Sigma and DIFCO catalogs.

Preferred cell culture conditions also relate to cell culture media thatinclude one or more antibiotics. Antibiotics suitable for use in cellculture media are well known in the art. See, e.g., Culture of AnimalCells: a manual of basic techniques (3^(rd) edition), 1994, Freshney(ed.), Wiley-Liss, Inc.; Cells: a laboratory manual (vol. 1), 1998),Spector, Goldman, Leinwand (eds.), Cold Spring Harbor Laboratory Press;and Animal Cells: culture and media, 1994, Darling & Morgan, John Wileyand Sons, Ltd., each of which is incorporated herein by reference in itsentirety including all figures, tables, and drawings.

Another example of a cell culture condition is a cell culture mediumthat contains one or more receptor ligands. Examples of receptor ligandsare well known to a person of ordinary skill in the art. Cytokinesand/or growth factors are preferred receptor ligands of the invention.See, e.g., R&D Systems Catalog, 614 McKinley Place N.E., Minneapolis,Minn. 55413. In exemplary embodiments, varying amounts of humanrecombinant leukemia inhibitory factor (hrLIF) and basic bovinefibroblast growth factor (bFGF) can be added to the culture medium toreprogram the precursor cells into totipotent cells. Varyingconcentrations of these two cytokines can be added to the culturemedium, preferably in concentrations of 1-1000 ng/mL, more preferably inconcentrations between 10-500 ng/mL, and most preferably about 100ng/mL. Exogenous soluble and membrane-associated forms of steel factorare not required for converting precursor cells into totipotent cells.

These examples are not meant to be limiting and any cytokine orcombination of cytokines can be added or deleted from those described inexemplary embodiments described hereafter. Preferred cytokines forgenerating totipotent cells can be selected from the group consisting offibroblast growth factor (FGF), leukemia inhibitor factor (LIF),cardiotrophin 1 (CT-1), ciliary neurotrophic factor (CNTF), stem cellfactor (SCF), oncostatin M (OSM), and any member of the interleukin (IL)family, including IL-6, IL-11, and IL-12.

Other cytokines and other molecules besides cytokines can be added ordeleted from the receptor ligand cocktail described in the exemplaryembodiments described hereafter to establish totipotent cells from anyof the cells described in the previous paragraph. Any of the conditionsfor generating totipotent cells can be modified from those describedherein. The ability of these modified conditions to generate totipotentcells can be monitored by methods defined in the section “IdentificationTotipotent Cells” described hereafter.

B. Identifying Totipotent Cells

Totipotent cells can be identified in a number of manners. Examples oftests for cell totipotency include:

(1) identifying a marker specific for totipotent cells;

(2) performing one or more nuclear transfer cycles with a cell (asdescribed hereafter) and developing the resulting embryo into an animal.

Markers can be utilized to distinguish totipotent cells fromnon-totipotent cells. Markers can be selected from the group of lowmolecular weight markers, macromolecular markers, cell surface markers,and intracellular markers. Examples of markers that may be suitable foridentifying totipotent cells can be selected from the group consistingof alkaline phosphatase, cytokeratin, vimentin, laminin, and c-kit.These markers are well known to a person of ordinary skill in the artand these examples are not meant to be limiting.

Some of these markers have been tested for cultured bovine cells beingidentified for totipotency. As noted previously, totipotent porcinecells of the invention may not appreciably stain for alkalinephosphatase. Therefore the cells of the invention are to be contrastedwith pluripotent cells discussed in previously referenced publications.It should be noted that some of the exemplary markers listed previouslymay not be specific for totipotent cells as some of these markers mayexist in pluripotent cells as well as in totipotent cells.

The invention relates to any markers specific for totipotent cells thatare known to a person of ordinary skill in the art. Markers fortotipotency that are not clearly defined in the art can be elucidated byprocesses such as differential display and genomics methods forelucidating totipotent cell markers. Totipotent cells may also beidentified by subjecting cells to analysis of nucleic acid sequencecontent (e.g., hybridization techniques with nucleic acid probes).Nucleic acid samples from totipotent cells and nucleic acid samples fromnon-totipotent cells can be screened for particular nucleic acidsequences. If samples from non-totipotent cells lack a nucleic acidsequence present in totipotent cells, then this nucleic acid sequencecould be a marker for distinguishing totipotent cells fromnon-totipotent cells. Similarly, if samples from non-totipotent cellsharbor a nucleic acid sequence that totipotent cells lack, this nucleicacid sequence could be a marker for distinguishing totipotent cells fromnon-totipotent cells. Similar methods can elucidate polypeptide markersby utilizing polypeptide analytical techniques (e.g., PAGE, SDS-PAGE,procedures comprising antibodies, and HPLC techniques known in the art).

A preferred test for cell totipotency is determining whether cells giverise to totipotent embryos and eventually cloned animals. This testrepresents a definitive test for cellular totipotency. An example ofsuch a test includes the following steps: (1) utilizing a potentiallytotipotent cell for nuclear transfer with an enucleated oocyte; (2)allowing the resulting cybrid to develop; (3) separating an embryo thatdeveloped from the cybrid into individual cells and subjecting one ormore of the individual cells to a second round of nuclear transfer; (4)allowing a resulting cybrid from step (3) to develop into an embryo; (5)implanting the embryo from step (2) or (4) into a uterine environment;and (6) allowing the embryo to develop. If the ensuing fetus developspast the first trimester of pregnancy then the cells initially used fornuclear transfer are most likely totipotent cells. If the cells utilizedfor nuclear transfer develop into a live born cloned animal then thecells are definitively totipotent. Examples of the techniques utilizedfor this exemplary test (e.g., enucleation of oocytes and nucleartransfer) are described completely in the art and in exemplaryembodiments defined hereafter.

Using the tests for identifying totipotent cells, the materials anddescribed herein can be modified by a person of ordinary skill in theart to produce totipotent cells from any type of precursor cell. Hence,the invention covers any of the materials and methods described hereinas well as modifications to these methods for generating totipotentcells, since a person of ordinary skill in the art can readily producetotipotent cells by utilizing the materials and methods described hereinin conjunction with methods for identifying totipotent cells.

C. Identifying Totipotent Cells that are Permanent Cells

The materials and methods described above (e.g., culturing the cellswith cytokines) may convert non-permanent cells into permanent cells.Other methods exist in the art for generating permanent cell lines fromprimary cells and for identifying permanent cells. For example,manipulating the activity of telomerase within the cells can immortalizecells. See, e.g., U.S. Pat. No. 5,645,986, entitled “Therapy andDiagnosis of Conditions Related to Telomere Length and/or TelomeraseActivity,” West et al., issued Jul. 8, 1997, and hereby incorporated byreference herein in its entirety including all figures, drawings, andtables.

Permanent cells can be identified by determining a number of times thatcultured cells undergo cell division and double in cell numbers beforethe cells terminate. As discussed above, permanent cells may double over10 times, 20 times, 30 times, 40 times, 50 times, and 60 times beforethe cells terminate. Materials and methods for measuring celltermination are taught above.

In addition, permanent cells can be identified by detecting the presenceand/or absence of low molecular weight and macromolecular markers thatare specific for permanent cells. The presence or lack of existence of amarker can be a determination of cell immortalization. In addition, aphenomenon associated with a marker can be an indication of immortality.For example, if the marker is an enzyme, an indication of the presenceof the enzyme and/or a certain level of catalytic activity of thatenzyme may be a suitable indication that a certain cell is permanent.

Low molecular weight markers include specific nucleosides, lipidassociated sialic acids, polyamines, and pseudouridine. These examplesare not limiting and the invention relates to any other low molecularweight markers known in the art.

Macromolecular markers can be separated into several classes includingnucleic acid polymers, peptides, polypeptides, proteins, enzymes, growthfactors, growth factor receptors, hormones, hormone receptors,oncogenes, oncogene products, and specific glycoproteins. Macromolecularmarkers can be selected from the group consisting of extracellularproteins, membrane associated proteins, and/or intracellular proteins,which may be membrane associated or soluble. One such marker forpermanent cells is telomerase or its associated activity, for example.See, U.S. Pat. No. 5,645,986, supra. Other examples of markers specificfor permanent cells can be selected from the following list. Theseexamples are not limiting and the invention relates to any markersspecific for permanent cells that are known in the art.

1) Epidermal growth factor (EGF) and its receptor (EGF-R)

2) Transforming growth factor-alpha (TGF-alpha) and its receptor

3) c-erbB2 receptor tyrosine kinase (HER2 product)

4) Hyaluronan receptor (probably CD44, an integral membraneglycoprotein)

5) Carcinoembryonic antigen (CEA) family of tumor markers (for exampleT1, a glycosylated protein)

6) Telomerase, a ribonucleoprotein which maintains telomere length inpermanent cells

7) Phosphatases: placental alkaline phosphatase (PLAP), germ cellalkaline phosphatase, prostate acid phosphatase (PAS)

8) Cathepsin D (catalyzes degradation of laminin).

9) Ornithine decarboxylase (ODC) (catalyzes the rate-limiting step inpolyamine synthesis)

10) Beta-glucuronidase

11) Alpha-6 integrin

12) Keratin K8

13) Oncogene products: ras oncogenes (k-ras, Ha-ras, p21), v-src, c-myc

14) Cyclin D1, cyclin A, and Retinoblastoma Gene Protein (Rb)

15) Changes in p53 expression or p53 mutations

16) Heterogeneous ribonucleoprotein-A2 (hnRNP-A2) over-expression

17) L-plastin

18) Ganglioside fucosyl-GM1

19) Mob-1 expression (mob-1) (homology to proinflammatory cytokines)

In addition to markers for cell permanence known in the art, markers forcell permanence can be identified using other methods well known in theart. For example, cell permanence markers can be identified by analyzingparticular molecules (e.g., nucleic acid molecules and polypeptidemolecules) that are unique to specific permanent cell types.

II. Transgenic Totipotent Porcine Cells

Materials and methods readily available to a person of ordinary skill inthe art can be utilized to convert totipotent porcine cells of theinvention into transgenic cells that are concomitantly totipotent. Oncethe nuclear DNA is modified in the totipotent cells of the invention,embryos and animals arising from these cells can lso comprise themodified nuclear DNA. Hence, materials and methods readily available toa person of ordinary skill in the art can be applied to the totipotentcells of the invention to produce transgenic animals and chimericanimals. See, e.g., EPO 264 166, entitled “Transgenic Animals SecretingDesired Proteins Into Milk”; WO 94/19935, entitled “Isolation ofComponents of Interest From Milk”; WO 93/22432, entitled “Method forIdentifying Transgenic Pre-implantation Embryos”; WO 95/17085, entitled“Transgenic Production of Antibodies in Milk;” Hammer et al., 1985,Nature 315: 680-685; Miller et al., 1986, J. Endocrinology 120: 481-488;Williams et al., 1992, J. Ani. Sci. 70: 2207-2111; Piedrahita et al.,1998, Biol. Reprod. 58. 1321-1329; Piedrahita et al., 1997, J. Reprod.Fert. (suppl.) 52: 245-254; and Nottle et al, 1997, J. Reprod. Fert.(suppl.) 52: 245-254, each of which is incorporated herein by referencein its entirety including all figures, drawings and tables.

Methods for generating transgenic cells typically include the steps of(1) assembling a suitable DNA construct useful for inserting a specificDNA sequence into the nuclear genome of a cell; (2) transfecting the DNAconstruct into the cells; (3) allowing random insertion and/orhomologous recombination to occur. The modification resulting from thisprocess may be the insertion of a suitable DNA construct(s) into thetarget genome; deletion of DNA from the target genome; and/or mutationof the target genome.

DNA constructs can comprise a gene of interest as well as a variety ofelements including regulatory promoters, insulators, enhancers, andrepressors as well as elements for ribosomal binding to the RNAtranscribed from the DNA construct. DNA constructs can also encoderibozymes and anti-sense DNA and/or RNA, identified previously herein.These examples are well known to a person of ordinary skill in the artand are not meant to be limiting.

Due to the effective recombinant DNA techniques available in conjunctionwith DNA sequences for regulatory elements and genes readily availablein data bases and the commercial sector, a person of ordinary skill inthe art can readily generate a DNA construct appropriate forestablishing transgenic cells using the materials and methods describedherein.

Transfection techniques are well known to a person of ordinary skill inthe art and materials and methods for carrying out transfection of DNAconstructs into cells are commercially available. For example, materialsthat can be used to transfect cells with DNA constructs are lipophilliccompounds, such as Lipofectin™, Superfect™, LipoTAXI™, and CLONfectin™.Particular lipophillic compounds can be induced to form liposomes formediating transfection of the DNA construct into the cells. In addition,cationic based transfection agents that are known in the art can beutilized to transfect cells with nucleic acid molecules (e.g., calciumphosphate precipitation). Also, electroporation techniques known in theart can be utilized to translocated nucleic acid molecules into cells.Furthermore, particle bombardment techniques known in the art can beutilized to introduce exogenous DNA into cells. Target sequences from aDNA construct can be inserted into specific regions of the nucleargenome by rational design of the DNA construct. These design techniquesand methods are well known to a person of ordinary skill in the art.See, U.S. Pat. No. 5,633,067, “Method of Producing a Transgenic Bovineor Transgenic Bovine Embryo,” DeBoer et al., issued May 27, 1997; U.S.Pat. No. 5,612,205, “Homologous Recombination in Mammalian Cells,” Kayet al., issued Mar. 18, 1997; and PCT publication WO 93/22432, “Methodfor Identifying Transgenic Pre-Implantation Embryos,” each of which isincorporated herein by reference in its entirety entirety, including allfigures, drawings, and tables. Once the desired DNA sequence is insertedinto the nuclear genome of a cell, the location of the insertion regionas well as the frequency with which the desired DNA sequence hasinserted into the nuclear genome can be identified by methods well knownto those skilled in the art.

Once a transgene or transgenes are inserted into the nuclear genome ofthe totipotent cell, that cell can be used as a nuclear donor forcloning a transgenic animal. A description of the embodiments related totransgenic animals are described in further detail hereafter.

A. Diseases and Parasites

Desired DNA sequences can be inserted into nuclear DNA of a cell toenhance the resistance of a cloned transgenic animal to particularparasites, diseases, and infectious agents. Examples of parasitesinclude worms, flies, ticks, and fleas. Examples of infectious agentsinclude bacteria, fungi, and viruses. Examples of diseases includeAtrophic rhinitis, Cholera, Leptospirosis, Pseudorabies, andBrucellosis. These examples are not limiting and the invention relatesto any disease or parasite or infectious agent known in the art. See,e.g., Hagan & Bruners Infectious Diesases of Domestic Animals (7thedition), Gillespie & Timoney, copyright 1981, Cornell University Press,Ithaca N.Y.

A transgene can confer resistance to a particular parasite or disease bycompletely abrogating or partially alleviating the symptoms of thedisease or parasitic condition, or by producing a protein which controlsthe parasite or disease.

B. Elements of DNA Constructs and Production of DNA Constructs

A wide variety of transcriptional and translational regulatory sequencesmay be employed, depending upon the nature of the host. Thetranscriptional and translational regulatory signals may be derived fromviral sources, such as adenovirus, bovine papilloma virus,cytomegalovirus, simian virus, or the like, whereas the regulatorysignals are associated with a particular gene sequence possessingpotential for high levels of expression. Alternatively, promoters frommammalian expression products, such as actin, casein, alpha-lactalbumin,uroplakin, collagen, myosin, and the like, may be employed.Transcriptional regulatory signals may be selected which allow forrepression or activation, so that expression of the gene product can bemodulated. Of interest are regulatory signals which can be epressed orinitiated by external factors such as chemicals or drugs. These xamplesare not limiting and the invention relates to any regulatory elements.ther examples of regulatory elements are described herein.

C. Examples of Preferred Recombinant Products

A variety of proteins and polypeptides can be encoded by a gene harboredwithin a DNA construct suitable for creating transgenic cells. Thoseproteins or polypeptides include hormones, growth factors, enzymes,clotting factors, apolipoproteins, receptors, drugs, pharmaceuticals,bioceuticals, nutraceuticals, oncogenes, tumor antigens, tumorsuppressors, cytokines, viral antigens, parasitic antigens, bacterialantigens and chemically synthesized polymers and polymers biosynthesizedand/or modified by chemical, cellular and/or enzymatic processes.Specific examples of these compounds include proinsulin, insulin, growthhormone, androgen receptors, insulin-like growth factor I, insulin-likegrowth factor II, insulin growth factor binding proteins, epidermalgrowth factor, TGF-α, TGF-β, dermal growth factor (PDGF), angiogenesisfactors (e.g., acidic fibroblast growth factor, basic fibroblast growthfactor, and angiogenin), angiogenesis inhibitors (e.g., endostatin andangiostatin), matrix proteins (Type IV collagen, Type VII collagen,laminin), oncogenes (ras,fos, myc, erb, src, sis, jun), E6 or E7transforming sequence, p53 protein, cytokine receptor, IL-1, IL-6, IL-8,IL-2, α, β, or γIFN, GMCSF, GCSF, viral capsid protein, and proteinsfrom viral, bacterial and parasitic organisms. Other specific proteinsor polypeptides which can be expressed include: phenylalaninehydroxylase, α-1-antitrypsin, cholesterol-7α-hydroxylase, truncatedapolipoprotein B, lipoprotein lipase, apolipoprotein E, apolipoproteinA1, LDL receptor, scavenger receptor for oxidized lipoproteins,molecular variants of each, VEGF, and combinations thereof. Otherexamples are monoclonal antibodies, antibody fragments, clottingfactors, apolipoproteins, drugs, tumor antigens, viral antigens,parasitic antigens, monoclonal antibodies, and bacterial antigens. Oneskilled in the art readily appreciates that these proteins belong to awide variety of classes of proteins, and that other proteins withinthese classes or outside of these classes can also be used. These areonly examples and are not meant to be limiting in any way.

It should also be noted that the genetic material which is incorporatedinto the cells from DNA constructs includes (1) nucleic acid sequencesnot normally present in target cells; (2) nucleic acid molecules whichare normally present in target cells but not expressed at physiologicalsignificant levels; (3) nucleic acid sequences normally present intarget cells and normally expressed at physiological desired levels; (4)other nucleic acid sequences which can be modified for expression intarget cells; and (5) any combination of the above.

In addition, DNA constructs may become incorporated into nuclear DNA ofcells, where incorporated DNA can be transcribed into ribonucleic acidmolecules that can cleave other RNA molecules at specific regions.Ribonucleic acid molecules which can cleave RNA molecules are referredto in the art as ribozymes. Ribozymes are themselves RNA molecules.Ribozymes can bind to discrete regions on a RNA molecule, and thenspecifically cleave a region within that binding region or adjacent tothe binding region. Ribozyme techniques can thereby decrease the amountof polypeptide translated from formerly intact message RNA molecules.

Furthermore, DNA constructs can be incorporated into nuclear DNA ofcells and when transcribed produce RNA that can bind to both specificRNA or DNA sequences. The nucleic acid sequences can be utilized inanti-sense techniques, which bind to the message (mRNA) and block thetranslation of these messages. Anti-sense techniques can thereby blockor partially block the synthesis of particular polypeptides in cells.

III. Nuclear Transfer

Nuclear transfer (NT) techniques using non-totipotent cells are wellknown to a person of ordinary skill in the art. See, e.g., U.S. Pat. No.4,664,097, “Nuclear Transplantation in the Mammalian Embryo byMicrosurgery and Cell Fusion,” issued May 12, 1987, McGrath & Solter;U.S. Pat. No. 4,994,384 (Prather et al.); and U.S. Pat. No. 5,057,420(Massey et al.), each of which is incorporated herein by reference inits entirety, including all figures, tables, and drawings. Exemplaryembodiments define a NT technique that may provide for efficientproduction of totipotent porcine embryos.

A. Nuclear Donors

Totipotent cells of the invention can be used as nuclear donors in a NTprocess for generating a cloned embryo. As described above, totipotentcells can be generated from nearly any type of cell. For NT techniques,a donor cell may be separated from a growing cell mass, isolated from aprimary cell culture, or isolated from a cell line. The entire cell maybe placed in the perivitelline space of a recipient oocyte or may bedirectly injected into the recipient oocyte by aspirating the nucleardonor into a needle, placing the needle into the recipient oocyte,releasing the nuclear donor and removing the needle withoutsignificantly disrupting the plasma membrane of the oocyte. Also, anucleus (e.g., karyoplast) may be isolated from a nuclear donor andplaced into the perivitelline space of a recipient oocyte or may beinjected directly into a recipient oocyte, for example.

B. Recipient Oocytes

A recipient oocyte is typically an oocyte with a portion of its ooplasmremoved, where the removed ooplasm comprises the oocyte nucleus.Enucleation techniques are well known to a person of ordinary skill inthe art. See e.g., Nagashima et al., 1997, Mol. Reprod. Dev. 48:339-343; Nagashima et al., 1992, J. Reprod. Dev. 38: 37-78; Prather etal., 1989, Biol. Reprod. 41: 414-418; Prather et al., 1990, J. Exp.Zool. 255: 355-358; Saito et al., 1992, Assis. Reprod. Tech. Andro. 259:257-266; and Terlouw et al., 1992, Theriogenology 37: 309, each of whichis incorporated herein by reference in its entirety including allfigures, tables, and drawings.

Oocytes can be isolated from either oviducts and/or ovaries of liveanimals by oviductal recovery procedures or transvaginal oocyte recoveryprocedures well known in the art and described herein. Furthermore,oocytes can be isolated from deceased animals. For example, ovaries canbe obtained from abattoirs and oocytes can be aspirated from theseovaries. The oocytes can also be isolated from the ovaries of a recentlysacrificed animal or when the ovary has been frozen and/or thawed.

Oocytes can be matured in a variety of media well known to a person ofordinary skill in the art. One example of such a medium suitable formaturing oocytes is depicted in an exemplary embodiment describedhereafter. Oocytes can be successfully matured in this type of mediumwithin an environment comprising 5% CO₂ at 39° C. Oocytes may becryopreserved and then thawed before placing the oocytes in maturationmedium. Cryopreservation procedures for cells and embryos are well knownin the art as discussed herein.

Components of an oocyte maturation medium can include molecules thatarrest oocyte maturation. Examples of such components are6-dimethylaminopurine (DMAP) and isobutylmethylxanthine (IBMX). IBMX hasbeen reported to reversibly arrest oocytes, but the efficiencies ofarrest maintenance are quite low. See, e.g., Rose-Hellkant and Bavister,1996, Mol. Reprod. Develop. 44: 241-249. However, oocytes may bearrested at the germinal vesicle stage with a relatively high efficiencyby incubating oocytes at 31° C. in an effective concentration of IBMX.Preferably, oocytes are incubated the entire time that oocytes arecollected. Concentrations of IBMX suitable for oocyte maturation are0.01 mM to 20 mM IBMX, preferably 0.05 mM to 10 mM IBMX, and morepreferably about 0.1 mM IBMX to about 0.5 mM IBMX, and most preferably0.1 mM IBMX to 0.5 mM IBMX.

A nuclear donor cell and a recipient oocyte can arise from the samespecie or different species. For example, a totipotent porcine cell canbe inserted into a porcine enucleated oocyte. Alternatively, atotipotent wild boar cell can be inserted into a domesticated porcineoocyte. Any nuclear donor/recipient oocyte combinations are envisionedby the invention. Preferably the nuclear donor and recipient oocyte fromthe same specie. Cross-species NT techniques can be utilized to producecloned animals that are endangered or extinct.

Oocytes can be activated by electrical and/or non-electrical meansbefore, during, and/or after a nuclear donor is introduced to recipientoocyte. For example, an oocyte can be placed in a medium containing oneor more components suitable for non-electrical activation prior tofusion with a nuclear donor. Also, a cybrid can be placed in a mediumcontaining one or more components suitable for nonelectrical activation.Activation processes are discussed in greater detail hereafter.

C. Injection/Fusion

A nuclear donor can be translocated into an oocyte using a variety ofmaterials and methods that are well known to a person of ordinary skillin the art. In one example, a nuclear donor may be directly injectedinto a recipient oocyte. This direct injection can be accomplished bygently pulling a nuclear donor into a needle, piercing a recipientoocyte with that needle, releasing the nuclear donor into the oocyte,and removing the needle from the oocyte without significantly disruptingits membrane. Appropriate needles can be fashioned from glass capillarytubes, as defined in the art and specifically by publicationsincorporated herein by reference.

In another example, at least a portion of plasma membrane from a nucleardonor and recipient oocyte can be fused together by utilizing techniqueswell known to a person of ordinary skill in the art. See, Willadsen,1986, Nature 320:63-65, hereby incorporated herein by reference in itsentirety including all figures, tables, and drawings. Typically, lipidmembranes can be fused together by electrical and chemical means, asdefined previously and in other publications incorporated herein byreference.

Examples of non-electrical means of cell fusion involve incubatingcybrids in solutions comprising polyethylene glycol (PEG), and/or Sendaivirus. PEG molecules of a wide range of molecular weight can be utilizedfor cell fusion.

Processes for fusion that are not explicitly discussed herein can bedetermined without undue experimentation. For example, modifications tocell fusion techniques can be monitored for their efficiency by viewingthe degree of cell fusion under a microscope. The resulting embryo canthen be cloned and identified as a totipotent embryo by the same methodsas those previously described herein for identifying totipotent cells,which can include tests for selectable markers and/or tests fordeveloping an animal.

D. Activation

Methods of activating oocytes and cybrids are known to those of ordinaryskill in the art. See, U.S. Pat. No. 5,496,720, “Parthenogenic OocyteActivation,” Susko-Parrish et al., issued on Mar. 5, 1996, herebyincorporated by reference herein in its entirety including all figures,tables, and drawings.

Both electrical and non-electrical processes can be used for activatingcells (e.g., oocytes and cybrids). Although use of a non-electricalmeans for activation is not always necessary, non-electrical activationcan enhance the developmental potential of cybrids, particularly whenyoung oocytes are utilized as recipients.

Examples of electrical techniques for activating cells are well known inthe art. See, WO 98/16630, published on Apr. 23, 1998, Piedraheidra andBlazer, hereby incorporated herein in its entirety including allfigures, tables, and drawings, and U.S. Pat. Nos. 4,994,384 and5,057,420. Non-electrical means for activating cells can include anymethod known in the art that increases the probability of cell division.Examples of non-electrical means for activating a nuclear donor and/orrecipient can be accomplished by introducing cells to ethanol; inositoltrisphosphate (IP₃); Ca²⁺ ionophore and protein kinase inhibitors suchas 6-dimethylaminopurine; temperature change; protein synthesisinhibitors (e.g., cyclohexamide); phorbol esters such as phorbol12-myristate 13-acetate (PMA); mechanical techniques, thapsigargin, andsperm factors. Sperm factors can include any component of a sperm thatenhance the probability for cell division. Other non-electrical methodsfor activation include subjecting the cell or cells to cold shock and/ormechanical stress.

Examples of preferred protein kinase inhibitors are protein kinase A, G,and C inhibitors such as 6-dimethylaminopurine (DMAP), staurosporin,2-aminopurine, sphingosine. Tyrosine kinase inhibitors may also beutilized to activate cells.

Activation materials and methods that are not explicitly discussedherein can be identified by modifying the specified conditions definedin the exemplary protocols described hereafter and in U.S. Pat. No.5,496,720.

Activation efficiency and totipotency that result from any modificationsof activation procedures can be identified by methods describedpreviously in the section entitled “Identification of Totipotent Cells.”Methods for identifying totipotent embryos can include one or moretests, such as (a) identifying specific markers for totipotent cells inembryos, and (b) by determining whether the embryos are totipotent byallowing them to develop into an animal. Therefore, the inventionrelates to any modifications to the activation procedures describedherein even though these modifications may not be explicitly statedherein.

F. Manipulation of Embryos Resulting from Nuclear Transfer

An embryo resulting from a NT process can be manipulated in a variety ofmanners. The invention relates to cloned embryos that arise from atleast one NT. Exemplary embodiments of the invention demonstrate thattwo or more NT procedures may enhance the efficiency for the productionof totipotent embryos. Exemplary embodiments indicate that incorporatingtwo or more NT procedures into methods for producing cloned totipotentembryos may enhance placental development. In addition, increasing thenumber of NT cycles involved in a process for producing totipotentembryos may represent a necessary factor for converting non-totipotentcells into totipotent cells. An effect of incorporating two or more NTcycles upon totipotency of resulting embryos is a surprising result,which was not previously identified or explored in the art.

Incorporating two or more NT cycles into methods for cloned totipotentembryos can provide further advantages. Incorporating multiple NTprocedures into methods for establishing cloned totipotent embryosprovides a method for multiplying the number of cloned totipotentembryos.

When multiple NT procedures are utilized for the formation of a clonedtotipotent embryo, oocytes that have been matured for any period of timecan be utilized as recipients in the first, second or subsequent NTprocedures. For example, if a first NT and then a second NT areperformed, the first NT can utilize an oocyte that has been matured forabout 53 hours as a recipient and the second NT may utilize an oocytethat has been matured for less than about 53 hours as a recipient.Alternatively, the first NT may utilize an oocyte that has been maturedfor about 53 hours as a recipient and the second NT may utilize anoocyte that has been matured for greater than about 53 hours as arecipient for a two-cycle NT regime. In addition, both NT cycles mayutilize oocytes that have been matured for about 53 hours as recipients,both NT cycles may utilize oocytes that have been matured for less thanabout 53 hours as recipients, and both NT cycles may utilize oocytesthat have been matured for greater than about 53 hours as recipients ina two-cycle NT regime.

For NT techniques that incorporate two or more NT cycles, one or more ofthe NT cycles may be preceded, followed, and/or carried outsimultaneously with an activation step. As defined previously herein, anactivation step may be accomplished by electrical and/or non-electricalmeans as defined herein. Exemplified embodiments described hereafterdescribe NT techniques that incorporate an activation step after one NTcycle. However, an activation step may also be carried out at the sametime as a NT cycle (e.g., simultaneously with the NT cycle) and/or anactivation step may be carried out prior to a NT cycle. Clonedtotipotent embryos resulting from a NT cycle can be (1) disaggregated or(2) allowed to develop further.

If embryos are disaggregated, disaggregated embryonic derived cells canbe utilized to establish cultured cells. Any type of embryonic cell canbe utilized to establish cultured cells. These cultured cells aresometimes referred to as embryonic stem cells or embryonic stem-likecells in the scientific literature. The embryonic stem cells can bederived from early embryos, morulae, and blastocyst stage embryos.Multiple methods are known to a person of ordinary skill in the art forproducing cultured embryonic cells. These methods are enumerated inspecific references previously incorporated by reference herein.

If embryos are allowed to develop into a fetus in utero, cells isolatedfrom that developing fetus can be utilized to establish cultured cells.In preferred embodiments, primordial germ cells, genital ridge cells,and fetal fibroblast cells can be isolated from such a fetus. Culturedcells having a particular morphology that is described herein can bereferred to as embryonic germ cells (EG cells). These cultured cells canbe established by utilizing culture methods well known to a person ofordinary skill in the art. Such methods are enumerated in publicationspreviously incorporated herein by reference and are discussed herein.

Cloned totipotent embryos resulting from NT can also be manipulated bycryopreserving and/or thawing the embryos. See, e.g., Nagashima et al.,1989, Japanese J. Anim. Reprod. 35: 130-134 and Feng et al., 1991,Theriogenology 35: 199, each of which is incorporated herein byreference in its entirety including all tables, figures, and drawings.Other embryo manipulation methods include in vitro culture processes;performing embryo transfer into a maternal recipient; disaggregatingblastomeres for NT processes; disaggregating blastomeres or inner cellmass cells for establishing cell lines for use in NT procedures; embryosplitting procedures; embryo aggregating procedures; embryo sexingprocedures; and embryo biopsying procedures. The exemplary manipulationprocedures are not meant to be limiting and the invention relates to anyembryo manipulation procedure known to a person of ordinary skill in theart.

IV. Development of Cloned Embryos

A. Identifying Totipotent Embryos

Totipotent embryos can be identified by the methods described in thesection “Identification of Totipotent Cells.” Individual cells can beisolated and subjected to similar tests. The tests relate to identifyingthe presence or absence of markers, for example. Also, a totipotentembryo can be identified by allowing an embryo to develop until itpasses the first trimester of gestation, or preferably, develops into alive born animal. Methods for identifying markers for totipotency arealso described herein.

B. Culture of Embryos In Vitro

Cloning procedures discussed herein provide an advantage of culturingcells and embryos in vitro prior to implantation into a recipientfemale. Methods for culturing embryos in vitro are well known to thoseskilled in the art. See, e.g., Nagashima et al., 1997, Mol. Reprod. Dev.48: 339-343; Petters & Wells, 1993, J. Reprod. Fert. (Suppl) 48: 61-73;Reed et al., 1992, Theriogenology 37: 95-109; and Dobrinsky et al.,1996, Biol. Reprod. 55:1069-1074, each of which is incorporated hereinby reference in its entirety, including all figures, tables, anddrawings. In addition, exemplary embodiments for media suitable forculturing cloned embryos in vitro are described hereafter. Feeder celllayers may or may not be utilized for culturing cloned embryos in vitro.Feeder cells are described previously and in exemplary embodimentshereafter.

C. Development of Embryos In Utero

Cloned embryos can be cultured in an artificial or natural uterineenvironment after NT procedures and embryo in vitro culture processes.Examples of artificial development environments are being developed andsome are known to those skilled in the art. Components of the artificialenvironment can be modified, for example, by altering the amount of acomponent or components and by monitoring the growth rate of an embryo.

Methods for implanting embryos into the uterus of an animal are alsowell known in the art, as discussed previously. Preferably, thedevelopmental stage of the embryo(s) is correlated with the estrus cycleof the animal.

Embryos from one specie can be placed into the uterine environment of ananimal from another specie. For example it has been shown in the artthat bovine embryos can develop in the oviducts of sheep. Stice &Keefer, 1993, “Multiple generational bovine embryo cloning,” Biology ofReproduction 48: 715-719. The invention relates to any combination of aporcine embryo in any other ungulate uterine environment. Across-species in utero development regime can allow for efficientproduction of cloned animals of an endangered species. For example, awild boar embryo can develop in the uterus of a domestic porcine sow.

Once an embryo is placed into the uterus of a recipient female, theembryo can develop to term. Alternatively, an embryo can be allowed todevelop in the uterus and then can be removed at a chosen time. Surgicalmethods are well known in the art for removing fetuses from uteri beforethey are born.

V. Cloned Porcine Animals

As described previously herein, the invention provides advantages ofbeing able to assess a phenotype of an animal before cloning thatanimal. Multiple products can be isolated from a cloned animal. Forexample, semen can be collected from a porcine animal, such as adomestic boar. Semen can be cryopreserved. Semen can also be separatedinto sex-specific fractions of sperm. See, U.S. Pat. Nos. 5,439,362,5,346,990, and 5,021,244, entitled “Sex-associated Membrane Proteins andMethods for Increasing the Probability that Offspring Will be of aDesired Sex,” Spaulding, and issued on Aug. 8, 1995, Sep. 13, 1994, andJun. 4, 1991, respectively, each of which is incorporated herein byreference in its entirety including all figures, drawings, and tables.Methods of collecting semen are well known to a person of ordinary skillin the art, as discussed previously.

In addition, the invention relates in part to any products collectedfrom a cloned porcine animal. The products can be any body fluids ororgans isolated from the animal, or any products isolated from thefluids or organs. In preferred embodiments, products such as meat may becollected from cloned porcine animals. In another embodiment, theinvention relates to determining the phenotype of a porcine animal,which is a neutered animal, and then cloning this animal such that thecloned animals are reproductively functional and can be used to producesemen. Other preferred embodiments of the invention relate to suchproducts as xenograft materials, sperm, embryos, oocytes, any type ofcells, and offspring harvested from cloned animals of the invention.

Xenograft materials, which are described previously herein, can relateto any cellular material extracted from one organism and placed intoanother organism. Medical procedures for extracting the cellularmaterial from one organism and grafting it into another organism arewell known to a person of ordinary skill in the art. Examples ofpreferable xenograft cellular materials can be selected from the groupconsisting of liver, lung, heart, nerve, gallbladder, and pancreascellular material.

As discussed in a previous section, transgenic animals can be generatedfrom the methods of the invention by using transgenic techniques wellknown to those of ordinary skill in the art. Preferably, clonedtransgenic porcine animals are produced from these methods. These clonedtransgenic animals can be engineered such that they are resistant orpartially resistant to diseases and parasites endemic to such animals.Examples of these diseases and parasites are outlined in a precedingsection.

Moreover, the cloned transgenic animals can be engineered such that theyproduce a recombinant product. Examples of recombinant products areoutlined in a preceding section. The expression of these products can bedirected to particular cells or regions within the cloned transgenicanimals by selectively engineering a suitable promoter element and otherregulatory elements to achieve this end.

For example, human recombinant products can be expressed in the urine ofcattle by operably linking a uroplakin promoter to the DNA sequenceencoding a recombinant product. Alternatively, examples are well knownto a person of ordinary skill in the art for selectively expressinghuman recombinant products in the milk of a bovine animal.

Once the recombinant product or products have been expressed in aparticular tissue or fluid of the cloned transgenic animal, the suitabletissue or fluid can be collected using methods well known in the art.Recombinant products can be purified from that fluid or tissue by usingstandard purification techniques well known to a person of ordinaryskill in the art.

EXAMPLES

The examples below are non-limiting and are merely representative ofvarious aspects and features of the present invention.

Example 1

Feeder Layer Preparation

A fetal fibroblast feeder cell layer was prepared from mouse fetusesthat were from 10 to 20 days gestation. The head, liver, heart andalimentary tract were removed and the remaining tissue washed andincubated at 37° C. in 0.05% trypsin and 0.53 mM EDTA (Gibco BRL catalogno. 15400-096). Loose cells were cultured in tissue culture dishescontaining MEM-alpha supplemented with penicillin, streptomycin, 10%fetal calf serum and 0.1 mM 2-mercaptoethanol. The feeder cell cultureswere established over a two to three week period at 37.4° C., 3.5% CO₂and humidified air. Before being used as feeder cells, mouse fibroblastswere pre-treated with mitomycin C (Calbiochem catalog no. 47589) at afinal concentration of 10 μg/ml for 3 hours and washed 5 times with PBSbefore pre-equilibrated growth media was added.

Feeder cells can be established from porcine fetuses as describedhereafter for establishing cultured porcine fetal fibroblast cells.

Example 2

Establishing Cultured Nuclear Donor Cells From Non-Embryonic Tissue

One advantage provided by the materials and methods defined herein isthe ability to establish a totipotent cell from virtually any type ofprecursor cell.

1. Establishing Cultured Cells from Porcine Fetal Tissue

Genital ridge cells were isolated from porcine fetuses and weredissociated with proteolytic enzymes (Pronase E or trypsin). Cellshaving EG cell morphology formed directly out of the genital ridgedigests but grew more quickly when established on mouse feeder layers(see, Example 1). The addition of growth factors hrLIF and bFGF to cellculture media had little effect on EG cell establishment andproliferation. EG cell proliferation rates increased when 17 mMD-glucose was incorporated into culture media. Cell culture mediacontained alpha-MEM, 10% fetal bovine serum, and 0.1 mM2-mercaptoethanol.

Fetuses of age 33, 36, 47 and 54 days were used as a source of genitalridge cells. EG cells from 33, 36 and 47 day old fetuses were moredifficult to establish and generally grew slowly. EG cells wereovergrown by other cell types, typically elongated cells which lookedlike fibroblasts, when cell cultures were established from genital ridgecells isolated from 33, 36 and 47 day old fetuses. In contrast, EG cellsfrom a 54 day old fetus grew relatively quickly and formed largecolonies within 1-2 weeks. EG cells were physically isolated from othercompeting cell types and were grown to confluence as a monolayer of oneapparent cell type with a typical EG cell morphology, a morphology thatis described herein.

2. Establishing Cultured Porcine Cumulus Cells

Cumulus cells were isolated from porcine cumulus-oocyte complexesmatured in vitro. Cumulus-oocyte complexes were collected fromslaughterhouse derived ovaries and matured in a porcine maturationmedium (described hereafter). Cumulus cells were stripped fromcumulus-oocyte complexes by placing the cumulus-oocyte complexes with500 μL TL HEPES in 15 ml-centrifuge tubes and vortexing the contents ofthe tubes using a vortex mixer (vortexed 3 minutes at half speed).

A cell suspension resulting from the vortexed cumulus-oocyte complexeswere transferred into a Petri dish after washing two times with 10 mL ofTL HEPES. The Petri dish was placed under a microscope and oocytes weremanually removed from the cell suspension with a small bore pipette.

Once oocytes were removed from the cell suspension, remaining cumuluscells were placed in a 15 mL centrifuge tube. The tube was placed in acentrifuge to pellet the cumulus cells. Pelleted cumulus cells wereresuspended in 5 mL of culture media containing MEM-alpha, 10% fetalcalf serum, 0.1 mM β-mercaptoethanol, and aliquoted into a 60 mm tissueculture dish at a cell density of approximately 25,000 cells/mL.

Cumulus cells were cultured using standard cell culture techniques. Cellculture media was changed every 3-4 days and cumulus cells were passagedwhen confluent.

3. Establishing Cultured Porcine Amniotic Cells

Amniotic fluid is collected from a pregnant porcine female using aguided needle technique. Amniotic fluid is transferred into 15 mLconical tubes and centrifuged at 1000×g for 10 minutes. Supernatantfluid is removed, leaving 1 mL of fluid on the cell pellet. The cellpellet is resuspended in that 1 mL of amniotic fluid. 0.5 mL of theamniotic cell sample is added to 4.5 ml of complete AmnioMax medium (90mL of AmnioMax-C100 Basal medium +15 mL of AmnioMax-C100 supplement[Gibco BRL]) in a T25 culture flask and placed in an incubator having atemperature of 39° C. and an atmosphere containing 5% CO₂.

Amniotic cells attach to culture flasks within approximately four days.Amniotic cell cultures were typically 70% confluent within 7-14 days.Cells are passaged by removing cell culture medium from flasks andwashing cells twice with EBSS media or PBS media (without calcium ormagnesium, Gibco BRL). 0.2 ml of a Trypsin/EDTA solution (Gibco BRL) isadded to culture flasks and incubated at 39° C. until cells releasedfrom culture flask surfaces. After cells release from culture flasksurfaces, cell culture media is added to flasks to inactivate trypsinand cells were divided over three new T25 flasks.

4. Establishing Cultured Porcine Fetal Fibroblast Cells

Cultured porcine fetal fibroblast cells were prepared from porcinefetuses that were from 33 to 54 days gestation. The head, liver, heartand alimentary tract were removed and the remaining tissue washed andincubated at 37° C. in 0.05% trypsin and 0.53 mM EDTA (Gibco BRL CatalogNo. 15400-096) diluted with Ca2+ and Mg2+ free Dulbecco's PBS (Gibco BRLCatalog No. 14190-151). Loose cells were cultured in tissue culturedishes containing MEM-alpha (Gibco BRL catalog no. 32561-037)supplemented with penicillin, streptomycin, 10% fetal bovine serum(Hyclone catalog no. SH30070.03), and 0.1 mM 2-mercaptoethanol (GibcoBRL catalog no. 21985-023). Porcine fetal cells were established over atwo to three week period at 37.4° C., 3.5% CO₂ and humidified air.

5. Establishing Cultured Porcine Fibroblast Cells from Adult Animals

A first step towards establishing totipotent cells from tissues of grownanimals is establishing a primary culture of isolated cells. Althoughthe following procedure relates to ear punch samples, this procedure canapply to cells isolated from any type of tissue. The following steps arepreferably performed utilizing sterile procedures:

1. Wash each ear sample twice with 2 mL of trypsin/EDTA solution in twoseparate 35 mm Petri dishes. Process each ear sample separately. Mincethe ear sample with sterile scissors and scalpel in a 35 mm Petri dishcontaining 2 mL of trypsin/EDTA solution. The minced pieces arepreferably less than 1 mm in diameter.

2. Incubate minced ear pieces in the trypsin/EDTA solution for 40-50min. in a 37° C. incubator with occasional swirling. The trypsin/EDTAsolution is described in more detail hereafter. The dish may be wrappedwith a stretchable material, such as Parafilm®, to reduce CO₂accumulation.

3. Transfer digested ear pieces to a 15 mL sterile tube. Wash the dishfrom which the digested ear pieces were recovered with 2 mL of thetrypsin/EDTA solution and transfer this wash solution to the steriletube.

4. Vortex the tube at high speed for 2 min.

5. Add 5 mL of cell culture media (defined below) to inactivate thetrypsin.

6. Centrifuge the 15 mL tube at 280×g for 10 minutes.

7. Decant the supernatant and re-suspend the cell pellet in residualsolution by gently taping the side of the tube.

8. Add 2 mL of cell culture media to the tube and then centrifuge asdescribed in step 6.

9. Decant the supernatant, re-suspend the pellet as described in step(7), then add 2 mL of media.

10. Keep 2-3 pieces of the ear for DNA analysis and store at −80° C. ina 2 mL tube.

11. Transfer resuspended cells into a 35 mm culture dish and incubate at37° C. in a humidified 5% CO₂/95% air atmosphere.

12. Change media every 2-4 days.

A trypsin/EDTA solution was prepared by mixing a trypsin-EDTA solution(Gibco BRL Catalog No. 15400-096) with Ca²⁺-free and Mg²⁺-freeDulbecco's phosphate-buffered saline (PBS) (Gibco BRL catalog no.14190-151) such that the solution contained 0.05% trypsin (w/v) and 0.53mM EDTA. The solution was sterilized by filtration through a 0.2 μmfilter.

A cell culture medium was prepared by combining minimum essentialMEM-alpha (Gibco BRL catalog no. 32561-037), 10% fetal bovine serum(Hyclone #SH30070.03), 0.1 mM 2-mercaptoethanol (Gibco BRL catalog no.21985-023), 100 U/mL penicillin, 100 μg/mL streptomycin, 0.25 μg/mLamphotercin B (Fungizone).

Example 3

Reprogramming Cultured Porcine Cells

If cultured cells established by the processes taught in Example 2 arenot clearly totipotent, cells can be reprogrammed by the followingprocess.

A genital ridge cell suspension (final concentration 100,000 cells/ml)is placed into a 35 mm Petri dish containing a murine primary embryonicfibroblast feeder layer. Culture media can be minimum essentialMEM-alpha (Gibco BRL catalog no. 32561-037), 10% fetal bovine serum(Hyclone catalog no. SH30070.03), 0.1 mM 2-mercaptoethanol (Gibco BRLcatalog no. 21985-023), 100 ng/ml human recombinant leukemia inhibitoryfactor (hrLIF; R&D System catalog no. 250-L), 100 ng/ml bovine basicfibroblast growth factor (bFGF; R&D System catalog no. 133-FB) at 37.5°C. and 5% CO₂. Exogenous steel factor (e.g., membrane associated steelfactor and soluble steel factor) need not be added to the culture media.

Culture medium supplemented with 100 ng/mL hrLIF and 100 ng/mL bFGF istypically incubated with precursor cells for ten to fourteen days.Supplemented medium is replaced with fresh supplemented medium every 2-4days. At any time in this culture process, EG cells may be observed.These EG cells can be isolated from the cell culture and culturedseparately to establish homogenous EG cell cultures.

To maintain EG cell lines, high density population cells are passagedevery week at a dilution ratio of 1:4 to 1:8. Cells are passaged byincubating them with a 0.05% trypsin—0.53 mM EDTA mixture and preparingnew cultures in fresh growth medium. The growth promoting capacity ofMEM-alpha media for totipotent cells can be enhanced by addinginsulin-transferrin, sodium selenite supplement, diluted to 1:100 (Sigmacatalog no. 11884) to the cell culture media. As a preventive measureagainst mycoplasma contamination, short term cultivation with tylosinetartrate (Sigma catalog no. T3151) is carried out. Before NT, cell linesare tested for presence of mycoplasma by PCR performed with DNA primersspecific for mycoplasma sequences (Stratagene catalog no. 302007).

Example 4

Oocyte Maturation

1. Oocyte Aspiration and Collection

Sow ovaries were transported to a laboratory at 25° C. and immediatelyashed with 0.9% saline. Follicles between 3-6 mm are aspirated using 18gauge needles and 50 mL tubes connected to a vacuum system, where thevacuum was adjusted for a flow rate of 45 mL/minute. Followingaspiration, oocytes were allowed to settle for 5-10 minutes. Follicularfluid (pFF) was aspirated and saved for use in maturation systems, ifneeded.

Oocytes were washed by resuspending them in 20 mL Hepes-PVA medium andthen allowing the oocytes to settle. The medium was removed and thewashing step was repeated two more times. Hepes-PVA medium contains 114mM NaCl, 3 mM KCL, 2 mM CaCl₂, 0.5 mM MgCl₂, 2 mM NaHCO₃, 0.3 mMNaH₂PO₄, 10 mM HEPES, 12 mM sorbitol, 0.2 mM sodium pyruvate, 005 μL/mLgentamycin, 0.07 grams/L penicillin G, 0.1 grams/L polyvinyl alcohol,and 2 mL/L sodium lactate and has a pH of 7.4. Washed oocytes were thenplaced into maturation media.

2. In Vitro Maturation

Oocytes were washed three times in maturation media and fiftyoocytes/well were transferred to 0.5 mL of maturation media in 4 wellplates for 20-22 hours, where the maturation media contained hormones.In addition, 6-8 follicle shell pieces were added to the wells, althoughthe addition of the shell pieces is optional. These follicle shellpieces are known to improve cytoplasmic maturation of porcine oocytes,which results in an increase in the developmental potential of theoocyte. See, e.g., Abeydeera et al., 1998, Biol. Reprod. 58: 213-218.

Maturation media contained 109 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 1 mMMgSO₄, 25 mM NaHCO₃, 1 mM KH₂PO₄, 6 mM glucose, 1 mM glutamine, 12 mMsorbitol, 5 mg/L insulin, 100 IU/L penicillin G, and 50 mg/Lstreptomycin and has a pH of 7.4. Porcine follicular fluid (pFF) can beadded to the maturation media in a concentration of approximately 10%(volume to volume). Also, β-mercaptoethanol can be added to thematuration media to a final concentration of 50 μM. In addition,cysteine can be added to the maturation media to a final concentrationof 0.6 mM. Maturation media with hormones contained 10 ng/mL epidermalgrowth factor, 10 U/mL equine chorionic gonadotropin, 10 U/mL humanchorionic gonadotropin, and 1 mM db-cAMP.

The oocytes and follicle shell pieces were then transferred to 0.5 mL offresh maturation media and matured for an additional 20-22 hours, wherematuration media did not contain hormones or db-cAMP. Oocytes werematured for a total of 40-44 hours at 39° C. in 5.0% CO₂ atmosphere.

Example 5

Nuclear Transfer

After 42-46 hours of maturation, oocytes were stripped of cumulus cellsby vortexing the matured oocytes for approximately 2 minutes. Aftervortexing, the oocytes were evaluated for polar body formation rates.Polar body formation rates can be evaluated by visual detection with astereomicroscope. Visual evaluation of polar body formation rates can beenhanced by staining oocytes in a medium containing 3 μg/mL Hoechst33342 and 7.5 μg/mL cytochalasin B for approximately 20 minutes. Oocyteswere then enucleated with an enucleation pipette.

NT processes were performed after oocytes were enucleated. In each NTprocedure, a nuclear donor cell was placed adjacent to an oocyte suchthat a portion of the plasma membrane for each cell touched one anotherand such that the nuclear donor was adjacent to the zona pellucida ofthe oocyte. Porcine EG cells, cultured porcine genital ridge cells,cultured porcine fibroblast cells, and cultured porcine cumulus cells,and cumulus cells that have been dissociated from a cumulus-oocytecomplex following 48 hour maturation were used as nuclear donors for theNT processes.

After a nuclear donor was placed adjacent to an enucleated oocyte, thetwo cells were fused. Three media can be used for fusing the cells: (1)0.25 M sorbitol, 0.1 mM CaOAc, 0.5 mM MgOAc, and 0.1% BSA; (2) 0.3 Mmannitol, 0.1 mM MgSO₄, 0.5 mM CaCl₂, and 0.1% BSA; (3) 0.3 M mannitol,5% TL Hepes, and 0.3% BSA; and (4) Zimmerman's medium. After fusion, thefused oocytes were placed in culture for 4 hours, where the culturecontained 109 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgSO₄, 25 mM NaHCO₃, 1mM KH₂PO₄, 6 mM glucose, 1 mM glutamine, 7 mM taurine, 5 mM hypotaurine,0.4% BSA, 100 IU/L penicillin G, and 50 mg/L streptomycin and had a pHof 7.4. The fused oocytes can also be cultured in a CR2 medium (CR1medium supplemented with amino acids), the latter of which is describedin U.S. Pat. No. 5,096,822, “Bovine embryo medium,” Rosenkrans Jr. etal., Nov. 3, 1992, hereby incorporated herein by reference in itsentirety, including all figures, tables, and drawings.

Example 6

Activation

After being cultured for 4 hours after fusion, fused oocytes wereincubated in 10 μM ionomycin in ZI solution (TL Hepes stock+1 mg/ml BSA)for 8 minutes and then rinsed in Z30 solution (TL Hepes stock+30 mg/mlBSA) for 5 minutes. TL Hepes stock contained 114 mM NaCl, 3 mM KCl, 2 mMNaHCO₃, 0.3 mM NaH₂PO₄, 10 mM Hepes, 2 mM CaCl₂, 0.5 mM MgCl₂, 2 ml/Lsodium lactate, and 1 ml/L phenol red and has a pH of 7.4

Oocytes were washed 3 times with TL Hepes PVA and then placed in 2 mMDMAP solution in CR2 medium with BSA for 4 hrs at 39° C. in a 5.0% CO₂atmosphere. Following activation, activated oocytes were rinsed threetimes with TL Hepes PVA and placed in porcine embryo culture media.

Example 7

In Vitro Culture of Activated Oocytes and Embryos

After removal from DMAP solution, activated oocytes and cybrids werewashed 3 times in TL Hepes PVA and placed in porcine embryo culturemedia for 4 days. Embryo culture media contained 109 mM NaCl, 5 mM KCl,2 mM CaCl₂, 1 mM MgSO₄, 25 mM NaHCO₃, 1 mM KH₂PO₄, 6 mM glucose, 1 mMglutamine, 12 mM sorbitol, 5 mg/L insulin, 100 IU/L penicillin G, and 50mg/L streptomycin and has a pH of 7.4. Activated ooyctes and cybrids mayalso be cultured in CR2 medium. On day 4, embryos are evaluated forcleavage and transferred into fresh embryo culture media that issupplemented with 10% fetal calf serum (v/v) for an additional 3 days.On day 7 embryos are evaluated for development to blastocysts.

Example 8

Recloning Process

One or more optional recloning cycles can be employed for enhancingtotipotency of nuclear donor cells. In a recloning cycle, embryos from afirst NT cycle are disaggregated either by pronase E (1-3 mg/ml in TLHEPES) or mechanically after treatment with cytochalasin B, when theembryos are at the morula stage. Single blastomeres are placed into theperivitelline space of an enucleated oocyte (52-54 hours postmaturation, as taught in Example 4).

A single blastomere is fused into the enucleated oocyte viaelectrofusion in a 500 μm chamber with an electrical pulse of 105V for15μs in an isotonic sorbitol solution (0.25 M) at 30° C. It is notrequired that the fused oocyte is subject to chemical activation forrecloning cycles.

After oocyte fusion, fused oocytes from the second NT cycle are culturedin embryo culture media under humidified air with 5% CO₂ at 39° C. Suchembryos can be transferred into a recipient female.

Example 9

Transfer of Porcine Embryos into a Maternal Recipient and Development ofa Cloned Porcine Animal

Ten gilts are injected with gonadotrophins (PG600™), which should resultin approximately 2-5 recipients displaying estrus 3-5 dayspost-treatment. Synchronized gilts will be utilized for embryo transfereither on day 3 or day 5 of estrus. Early stage embryos (<8 cells) aretransferred into recipients on day 3 of estrus, and later stage embryos(>8 cells) are transferred into additional recipients on day 5 ofestrus. Animals are anesthetized, a mid-ventral laparotomy performed andthe reproductive tract exposed. The embryo transfer procedure consistsof transferring 30-50 early stage embryos that will be deposited intothe oviduct, or transferring later stage embryos (morula and/orblastocyst stage embryos) into the uterine horn. Recipients areevaluated for initiation of pregnancy and are continually monitored withweekly ultrasound exams.

Example 10

Cloning Transgenic Porcine Animals

Transgenic cells suitable for establishing a cloned transgenic porcineanimal can be prepared from cells isolated from an adult animal. FIG. 3illustrates processes that can be utilized to establish such transgeniccells. Although transgenic cells can be established from nearly any celltype by using the teachings of the invention, FIG. 3 illustratesprocedures for establishing transgenic embryonic stem cells andtransgenic totipotent cells.

Fibroblast cell cultures can be established from ear punches extractedfrom a porcine animal as defined previously. In addition, culturedfibroblast cells can be established from porcine fetuses. Individualcells can be isolated from this cell culture and utilized as nucleardonors in a nuclear transfer process. A single nuclear transfer cycle ormultiple nuclear transfer cycles can be applied. Other optional stepsare defined in previous examples.

Cells can then be transfected with a DNA construct. Cells can betransfected at multiple steps, as indicated in FIG. 3. Materials andmethods for preparing transgenic cells are defined in publicationsreferenced previously. Totipotent cells of the invention can betransfected with a DNA comprising (a) an antibiotic resistance gene; (b)a DNA sequence encoding a protein or proteins; and (c) a promoterelement or elements. The transfected cells are selected for transgenicmodification by selection in cell culture media containing antibiotic.The transgenic cells are then screened for transgenic modification byutilizing one or more screening techniques. Examples of these techniquesare: (1) polymerase chain reaction, (2) Southern blotting, and (3)fiber-FISH procedures. These techniques are well known to a person ofordinary skill in the art. The latter two techniques can be utilized todetermine the number of copies of an inserted gene sequence in embryonicgerm cell nuclear DNA.

These screening procedures can be applied to transfected cells at any ofthe steps indicated in FIG. 3. Cloned transgenic animals are establishedby utilizing a transgenic cell as a nuclear donor in an NT process, asdescribed in Example 5. Fused oocytes produced by such a process aresubjected to the procedures taught in Examples 6, 7, and 9 andoptionally 8 for the production of a cloned transgenic porcine animal.

While the invention has been described and exemplified in sufficientdetail for those skilled in this art to make and use it, variousalternatives, modifications, and improvements should be apparent withoutdeparting from the spirit and scope of the invention.

One skilled in the art readily appreciates that the present invention iswell adapted to carry out the objects and obtain the ends and advantagesmentioned, as well as those inherent therein. The cell lines, embryos,animals, and processes and methods for producing them are representativeof preferred embodiments, are exemplary, and are not intended aslimitations on the scope of the invention. Modifications therein andother uses will occur to those skilled in the art. These modificationsare encompassed within the spirit of the invention and are defined bythe scope of the claims.

It will be readily apparent to a person skilled in the art that varyingsubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention.

All patents and publications mentioned in the specification areindicative of the levels of those of ordinary skill in the art to whichthe invention pertains. All patents and publications are hereinincorporated by reference to the same extent as if each individualpublication was specifically and individually indicated to beincorporated by reference.

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof” and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed bypreferred embodiments and optional features, modification and variationof the concepts herein disclosed may be resorted to by those skilled inthe art, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the appended claims.

In addition, where features or aspects of the invention are described interms of Markush groups, those skilled in the art will recognize thatthe invention is also thereby described in terms of any individualmember or subgroup of members of the Markush group. For example, if X isdescribed as selected from the group consisting of bromine, chlorine,and iodine, claims for X being bromine and claims for X being bromineand chlorine are fully described.

Other embodiments are set forth within the following claims.

What is claimed is:
 1. A method for preparing a porcine embryo capableof developing into a live-born porcine animal, the method comprising:(a) obtaining a totipotent porcine nuclear donor cell from a cellculture; (b) forming a cybrid by translocating said totipotent porcinenuclear donor cell, or nucleus thereof, into an enucleated porcineoocyte, wherein prior to enucleation, said oocyte has been matured forbetween 42 and 56 hours, and before, during or after said translocation,activating said oocyte; (c) culturing said cybrid to establish saidporcine embryo.
 2. A method according to claim 1, wherein said porcinenuclear donor cell is a transgenic cell.
 3. A method according to claim1, further comprising cryopreserving said porcine embryo.
 4. The methodaccording to claim 1 wherein said forming of step (b) comprises: (i)fusing said cultured totipotent porcine nuclear donor cell and saidenucleated oocyte to form a fused cell; and (ii) activating said fusedcell.
 5. A method according to claim 4, wherein said fusion of step (i)comprises using an electrical stimulus.
 6. A method according to claim4, wherein said activation of step (ii) comprises increasingintracellular levels of divalent cations and reducing phosphorylation ofcellular proteins in said fused cell.
 7. A method according to claim 6,wherein said activation of step (ii) comprises incubating said fusedcybrid in a medium comprising ionomycin and/or 6-dimethylaminopurine. 8.A method according to claim 7, wherein said medium comprises ionomycinat a concentration of from 10 μM to 50 μM.
 9. A method according toclaim 1, wherein said totipotent porcine nuclear donor cell is a fetalcell.
 10. A method according to claim 9, wherein said fetal cell is asomatic cell or a genital ridge cell.
 11. A method according to claim 1,wherein said totipotent porcine nuclear donor cell is not serum starved.12. A method according to claim 1, wherein said cell culture isincubated in a medium comprising greater than 10 mM glucose.
 13. Amethod according to claim 1, wherein said cell culture is incubated in amedium comprising one or more components selected from the groupconsisting of leukemia inhibitor factor, cardiotrophin 1, ciliaryneurotrophic factor, stem cell factor, oncostatin M, interleukin-6,interleukin-11, interleukin-12, and fibroblast growth factor.
 14. Aprocess for preparing a porcine animal, the method comprising: (a)obtaining a totipotent porcine nuclear donor cell from a cell culture;(b) forming a cybrid by translocating said totipotent porcine nucleardonor cell, or nucleus thereof, into an enucleated porcine oocyte,wherein prior to enucleation, said oocyte has been matured for between42 and 56 hours, and before, during or after said translocation,activating said oocyte; (c) culturing said cybrid to establish saidporcine embryo; and (d) transferring said porcine embryo into arecipient porcine female so as to produce a fetus that undergoes fullfetal development and parturition to generate a live-born porcineanimal.
 15. A method according to claim 14, wherein said totipotentnuclear donor cell is a transgenic cell.
 16. A method according to claim14, further comprising cryopreserving said porcine embryo.
 17. Themethod according to claim 1 wherein said forming of step (b) comprises:(i) fusing said totipotent porcine nuclear donor cell and saidenucleated oocyte to form a fused cell; and (ii) activating said fusedcell.
 18. A method according to claim 17, wherein said fusion of step(i) comprises using an electrical stimulus.
 19. A method according toclaim 17, wherein said activation of step (ii) comprises increasingintracellular levels of divalent cations and reducing phosphorylation ofcellular proteins in said fused cell.
 20. A method according to claim19, wherein said activation of step (ii) comprises incubating said fusedcell in a medium comprising ionomycin and/or 6-dimethylaminopurine. 21.A method according to claim 20, wherein said medium comprises ionomycinat a concentration of from 10 μM to 50 μM.
 22. A method according toclaim 14, wherein said totipotent porcine nuclear donor cell is a fetalcell.
 23. A method according to claim 22, wherein said fetal cell is asomatic cell or a genital ridge cell.
 24. A method according to claim14, wherein said totipotent porcine nuclear donor cell is not serumstarved.
 25. A method according to claim 14, wherein said methodcomprises one or more recloning steps.
 26. A method according to claim14, wherein said cell culture is incubated in a medium comprisinggreater than 10 mM glucose.
 27. A method according to claim 14, whereinsaid cell culture is incubated in a medium comprising one or morecomponents selected from the group consisting of leukemia inhibitorfactor, cardiotrophin 1, ciliary neurotrophic factor, stem cell factor,oncostatin M, interleukin-6, interleukin-11, interleukin-12, andfibroblast growth factor.
 28. A process for preparing a totipotentporcine fetus, the method comprising: (a) obtaining a totipotent porcinenuclear donor cell from a cell culture; (b) forming a cybrid bytranslocating said totipotent porcine nuclear donor cell, or nucleusthereof, into an enucleated porcine oocyte, wherein prior toenucleation, said oocyte has been matured for between 42 and 56 hours,and before, during or after said translocation, activating said oocyte;(c) culturing said cybrid to establish said porcine embryo; and (d)transferring said porcine embryo into a recipient porcine female so asto produce a totipotent porcine fetus.
 29. A method according to claim28, wherein said totipotent nuclear donor cell is a transgenic cell. 30.A method according to claim 28, further comprising cryopreserving saidporcine embryo.
 31. The method according to claim 1 wherein said formingof step (b) comprises: (i) fusing said totipotent porcine nuclear donorcell and said enucleated oocyte to form a fused cell; and (ii)activating said fused cell.
 32. A method according to claim 31, whereinsaid fusion of step (i) comprises using an electrical stimulus.
 33. Amethod according to claim 31, wherein said activation of step (ii)comprises increasing intracellular levels of divalent cations andreducing phosphorylation of cellular proteins in said fused cell.
 34. Amethod according to claim 33, wherein said activation of step (ii)comprises incubating said fused cell in a medium comprising ionomycinand/or 6-dimethylaminopurine.
 35. A method according to claim 34,wherein said medium comprises ionomycin at a concentration of from 10 μMto 50 μM.
 36. A method according to claim 28, wherein said totipotentporcine nuclear donor cell is a fetal cell.
 37. A method according toclaim 36, wherein said fetal cell is a somatic cell or a genital ridgecell.
 38. A method according to claim 28, wherein said totipotentporcine nuclear donor cell is not serum starved.
 39. A method accordingto claim 28, wherein said method comprises one or more recloning steps.40. A method according to claim 28, wherein said cell culture isincubated in a medium comprising greater than 10 mM glucose.
 41. Amethod according to claim 28, wherein said cell culture is incubated ina medium comprising one or more components selected from the groupconsisting of leukemia inhibitor factor, cardiotrophin 1, ciliaryneurotrophic factor, stem cell factor, oncostatin M, interleukin-6,interleukin-11, interleukin-12, and fibroblast growth factor.
 42. Aprocess for preparing a porcine animal, the method comprising: (a)obtaining a porcine nuclear donor cell from a cell culture, wherein saidcell is a fetal cell; (b) forming a cybrid by nuclear transfer of saidtotipotent porcine nuclear donor cell, or the nucleus thereof, into anenucleated oocyte, wherein said nuclear transfer comprises: (i) placingsaid porcine nuclear donor cell into the perivitelline space of anenucleated porcine oocyte; (ii) fusing said porcine nuclear donor celland said enucleated porcine oocyte to form a fused cell, wherein, priorto enucleation, said oocyte has been matured for between 42 and 56hours; and (iii) activating said fused cell to form said cybrid; (c)culturing said cybrid to establish a porcine embryo; and (d)transferring said porcine embryo into a recipient female so as toproduce a fetus that undergoes full fetal development and parturition togenerate said live-born porcine animal.
 43. A method according to claim42, wherein said fusion of in step (i) comprises an electrical stimulus,and said activation of step (ii) comprises increasing intracellularlevels of divalent cations and reducing phosphorylation of cellularproteins in said fused cell.
 44. A method according to claim 43, whereinsaid activation of step (ii) comprises incubating said fused cell in amedium comprising ionomycin and/or 6-dimethylaminopurine.
 45. A methodaccording to claim 12, wherein said medium comprises ionomycin at aconcentration of from 10 μM to 50 μM.
 46. A method according to claim43, wherein said cell culture is incubated in a medium comprisinggreater than 10 mM glucose.
 47. A method according to claim 43, whereinsaid cell culture is incubated in a medium comprising one or morecomponents selected from the group consisting of leukemia inhibitorfactor, cardiotrophin 1, ciliary neurotrophic factor, stem cell factor,oncostatin M, interleukin-6, interleukin-11, interleukin-12, andfibroblast growth factor.